Methods for treating Arenaviridae and Coronaviridae virus infections

ABSTRACT

Provided are methods for treating Arenaviridae and Coronaviridae virus infections by administering nucleosides and prodrugs thereof, of Formula I: 
                         
wherein the 1′ position of the nucleoside sugar is substituted. The compounds, compositions, and methods provided are particularly useful for the treatment of Lassa virus and Junin virus infections.

CROSS-REFERENCES TO RELATED APPLICATIONS

This patent application is a Continuation of U.S. patent applicationSer. No. 16/863,566, filed on Apr. 30, 2020, which is a Continuation ofU.S. patent application Ser. No. 16/265,016, filed on Feb. 1, 2019, nowU.S. Pat. No. 10,695,361, issued on Jun. 30, 2020, which is aContinuation of U.S. patent application Ser. No. 15/267,433, filed onSep. 16, 2016, now U.S. Pat. No. 10,251,904, issued on Apr. 9, 2019,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalPatent Application No. 62/219,302, filed Sep. 16, 2015 and U.S.Provisional Patent Application No. 62/239,696, filed Oct. 9, 2015. Theforegoing applications are incorporated herein by reference in theirentireties.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 1137P2C24_2021-04-05Sequence Listing.txt, date recorded: Apr. 5, 2021, size: 1 KB).

FIELD OF THE INVENTION

The invention relates generally to methods and compounds for treatingArenaviridae virus infections, particularly methods and nucleosides andprodrugs thereof for treating Lassa virus and Junin virus. The inventionrelates generally to methods and compounds for treating Coronaviridaevirus infections, particularly methods and nucleosides and prodrugsthereof for treating SARS virus and MERS virus.

BACKGROUND OF THE INVENTION

Lassa virus is a segmented negative-sense RNA virus that belongs to thefamily Arenaviridae. Arenaviruses are further sub-divided into the OldWorld and New World virus complexes based on serologicalcross-reactivity, phylogenetic relations, and geographical distribution,(Wulff, 1978; Bowen, 1997). The New World arenavirus complex comprisesviruses that circulate in North America (i.e., Whitewater Arroyo (WWAV),Tamiami (TAMV), and Bear Canyon (BCNV) viruses) and South America (i.e.,Tacaribe (TACV), Junin (JUNV), Machupo (MACV), Guanarito (GTOV), andSabia (SABV) viruses). The Old World complex includes arenaviruses thatcirculate in Africa, Europe, and Asia (i.e., lymphocyticchoreomeningitis (LCMV) and Lassa (LASV) viruses). The range ofreservoir rodent species restricts the geographic occurrence ofarenaviruses, with the exception of LCMV that is distributed worldwidedue to its association with Mus domesticus and M. musculus, which havemigrated globally (Salazar-Bravo, 2002). The reservoir hosts of LASV arerodents of the genus Mastomys that are enzootic in sub-Saharan Africa(Salazar-Bravo, 2002). At least seven arenaviruses are known to causesevere hemorrhagic fever in humans, among which are LASV, JUNV, MACV,GTOV, and SABV that are endemic in West Africa, Argentina, Bolivia,Venezuela, and Brazil, respectively, and recently discovered Lujo (LUJV)and Chapare (CHAPV) viruses that originated in Zambia and Bolivia,respectively (Breise, 2009; Delgado, 2008).

Lassa virus (LASV) is endemic to West Africa with an estimated300,000-500,000 people infected annually (McCormick, 1987). Transmissionoccurs through contact with infected rodents (Mastomys natalensis) orvirus-contaminated rodent excreta, and person-to-person transmission,especially in hospital settings, has been documented (McCormick, 1987).Disease caused by LASV ranges from subclinical infection to mild tosevere hemorrhagic fever that is associated with multi-organ failure.Mortality rates associated with LASV infection vary and range fromapproximately 2% to 15% for hospitalized cases and can exceed 50% incertain outbreak scenarios (McCormick, 1987; Fisher-Hoch, 1995). Despitethe high incidence and associated morbidity and mortality, there is noapproved therapy to treat LASV infection in humans. Supportive care andearly administration of ribavirin are current standard of care.

LASV initially infects monocytes, macrophages, and dendritic cells andspreads systemically to produce a primary viremia that leads toinfection of internal organs. Virus replication leads to a rise ininflammatory cytokine levels and development of coagulopathies resultingin vascular leakage, hypovolemic shock and multi-organ failure (Hensley,2011).

Replication of arenaviruses is catalyzed by the L polymerase proteinthat utilizes viral RNA templates that consist of genomic RNAencapsidated by the viral nucleocapsid protein NP and comprises viralribonucloprotein (RNP) (Buchmeier, 2007). Replication is initiated uponviral entry into the host cell where the L polymerase, associated withthe viral RNP, initiates transcription from the genome promoter locatedat the 3′-end of each genomic RNA segment, L and S. The primarytranscription event results in the synthesis of NP and L polymerase mRNAencoded in antigenomic orientation from the S and L segments,respectively. Transcription terminates at the distal side of thestem-loop (SL) structure within the intergenomic region (IGR).Arenaviruses utilize a cap snatching strategy to acquire the capstructures of cellular mRNAs to facilitate translation. Cap snatching ismediated by the endonuclease activity of the L polymerase that isco-factored by the cap binding activity of NP to produce cappednon-polyadenylated mRNAs. Subsequently, the L polymerase adopts areplicase mode and moves across the IGR to generate a full-lengthcomplementary antigenomic RNA (agRNA). This agRNA serves as a templatefor the synthesis of GPC and Z mRNAs encoded in genomic orientation fromthe S and L segments, respectively, and for the synthesis of full-lengthgenomic RNA (gRNA) (Buchmeier, 2007; Franze-Fernandez, 1987; Meyer,1993; Qi, 2010; Lelke, 2010; Morin, 2010).

Human coronaviruses, first identified in the mid-1960s, are commonviruses that infect most people at some time in their life, generallycausing mild to moderate upper respiratory and gastrointestinal tractillnesses. The novel coronavirus referred to as “Middle East RespiratorySyndrome Coronavirus” (MERS-CoV or MERS) was first reported in SaudiArabia in 2012 and has spread to several other countries. SARS-CoV, thecoronavirus responsible for Severe Acute Respiratory Syndrome (SARS) wasfirst recognized in China in 2002 and led to a worldwide outbreak in2002 and 2003.

SUMMARY OF THE INVENTION

Provided are methods and compounds for the treatment of infectionscaused by the Arenaviridae virus family.

Provided is a method for treating an Arenaviridae infection in a humanin need thereof comprising administering a therapeutically effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein:    -   each R¹ is H or halogen;    -   each R², R³, R⁴ or R⁵ is independently H, OR^(a), N(R^(a))₂, N₃,        CN, NO₂, S(O)_(n)R^(a), halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or        (C₂-C₈)substituted alkynyl;        -   or any two R², R³, R⁴ or R⁵ on adjacent carbon atoms when            taken together are —O(CO)O— or when taken together with the            ring carbon atoms to which they are attached form a double            bond;    -   R⁶ is OR^(a), N(R^(a))₂, N₃, CN, NO₂, S(O)₂R^(a), —C(═O)R¹¹,        —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹, —S(O)R¹¹, —S(O)₂R¹¹,        —S(O)(OR¹¹), —S(O)₂(OR¹¹), —SO₂NR¹¹R¹², halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl,        (C₂-C₈)substituted alkynyl, or (C₆-C₂₀)aryl(C₁-C₈)alkyl;    -   R⁷ is selected from a group consisting of        -   a) H, —C(═O)R¹¹, —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹,            —S(O)R¹¹, —S(O)₂R¹¹, —S(O)(OR¹¹), —S(O)₂(OR¹¹), or            —SO₂NR¹¹R¹²,            -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl,                (C₂-C₈)alkynyl or (C₆-C₂₀)aryl(C₁-C₈)alkyl of each R¹¹                or R¹² is, independently, optionally substituted with                one or more halo, hydroxy, CN, N₃, N(R^(a))₂ or OR^(a);                and wherein one or more of the non-terminal carbon atoms                of each said (C₁-C₈)alkyl may be optionally replaced                with —O—, —S— or —NR^(a)—,

-   -   -   -   wherein:                -   R^(c) is selected from phenyl, 1-naphthyl,                    2-naphthyl,

-   -   -   -   -   R^(d) is H or CH₃;                -   R^(e1) and R^(e2) are each independently H,                    (C₁-C₆)alkyl or benzyl;                -   R^(f) is selected from H, (C₁-C₈)alkyl, benzyl,                    (C₃-C₆)cycloalkyl, and —CH₂(C₃-C₆)cycloalkyl;                -   R^(g) is selected from (C₁-C₈)alkyl, —O(C₁-C₈)alkyl,                    benzyl, —O-benzyl, —CH₂—(C₃-C₆)cycloalkyl,                    —O—CH₂—(C₃-C₆)cycloalkyl, and CF₃; and                -   n′ is selected from 1, 2, 3, and 4; and

        -   d) a group of the formula:

-   -   -   -   wherein:                -   Q is O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;                -   Z¹ and Z², when taken together, are                    —Q¹(C(R^(Y))₂)₃Q¹-;                -   wherein                -    each Q¹ is independently O, S, or NR; and                -    each R^(y) is independently H, F, Cl, Br, I, OH, R,                    —C(═Q²)R, —C(═Q²)OR, —C(═Q²)N(R)₂, —N(R)₂, —⁺N(R)₃,                    —SR, —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR),                    —OC(═Q¹)R, —OC(═Q²)OR, —OC(═Q²)(N(R)₂), —SC(═Q²)R,                    —SC(═Q²)OR, —SC(═Q²)(N(R)₂), —N(R)C(═Q²)R,                    —N(R)C(═Q²)OR, —N(R)C(═Q²)N(R)₂, —SO₂NR₂, —CN, —N₃,                    —NO₂, —OR, or Z³; or when taken together, two R^(y)                    on the same carbon atom form a carbocyclic ring of 3                    to 7 carbon atoms;                -    each Q² is independently, O, S, NR, ⁺N(O)(R),                    N(OR), ⁺N(O)(OR), or N—NR₂; or                -   Z¹ and Z² are each, independently, a group of the                    Formula Ia:

-   -   -   -   -   wherein:                -    each Q³ is independently a bond, O, CR₂, NR,                    ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S, S—S, S(O), or                    S(O)₂;                -    M2 is 0, 1 or 2;                -    each R^(x) is independently R^(y) or the formula:

-   -   -   -   -    wherein:                -    each M1a, M1c, and M1d is independently 0 or 1;                -    M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;                -    Z³ is Z⁴ or Z⁵;                -    Z⁴ is R, —C(Q²)R^(y), —C(Q²)Z⁵, —SO₂R^(y), or                    —SO₂Z⁵; and                -    Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is                    independently substituted with 0 to 3 R^(y) groups;

    -   R⁸ is halogen, NR¹¹R¹², N(R¹¹)OR¹¹, NR¹¹NR¹¹R¹², N₃, NO, NO₂,        CHO, CN, —CH(═NR¹¹), —CH═NNHR¹¹, —CH═N(OR¹¹), —CH(OR¹¹)₂,        —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹², —C(═O)OR¹¹, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl,        (C₆-C₂₀)aryl(C₁-C₈)alkyl, OR¹¹ or SR¹¹;

    -   each R⁹ or R¹⁰ is independently H, halogen, NR¹¹R¹², N(R¹¹)OR¹¹,        NR¹¹NR¹¹R¹², N₃, NO, NO₂, CHO, CN, —CH(═NR¹¹), —CH═NHNR¹¹,        —CH═N(OR¹¹), —CH(OR¹¹)₂, —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹²,        —C(═O)OR¹¹, R¹¹, OR¹¹ or SR¹¹;

    -   each R¹¹ or R¹² is independently H, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl; or R¹¹ and R¹² taken together with a        nitrogen to which they are both attached form a 3 to 7 membered        heterocyclic ring wherein any one carbon atom of said        heterocyclic ring can optionally be replaced with —O—, —S—or        —NR^(a)—;

    -   each R^(a) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, —C(═O)R, —C(═O)OR, —C(═O)NR₂, —C(═O)SR,        —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), or —SO₂NR₂; wherein

    -   each R is independently H, (C₁-C₈) alkyl, (C₁-C₈) substituted        alkyl, (C₂-C₈)alkenyl, (C₂-C₈) substituted alkenyl, (C₂-C₈)        alkynyl, (C₂-C₈) substituted alkynyl, (C₆-C₂₀)aryl,        (C₆-C₂₀)substituted aryl, (C₂-C₂₀)heterocyclyl,        (C₂-C₂₀)substituted heterocyclyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl or        substituted (C₆-C₂₀)aryl(C₁-C₈)alkyl;

    -   each n is independently 0, 1, or 2; and

    -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl of each R², R³, R⁵, R⁶, R¹¹ or R¹² is,        independently, optionally substituted with one or more halo,        hydroxy, CN, N₃, N(R^(a))₂ or OR^(a); and wherein one or more of        the non-terminal carbon atoms of each said (C₁-C₈)alkyl may be        optionally replaced with —O—, —S—or —NR^(a)—.

In another embodiment, the method comprises administering atherapeutically effective amount of a racemate, enantiomer,diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form,hydrate or solvate of a compound of Formula I or a pharmaceuticallyacceptable salt or ester thereof to a mammal in need thereof.

In another embodiment, the method comprises treating an Arenaviridaeinfection in a human in need thereof by administering a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt or ester thereof.

In another embodiment, the method comprises treating a Lassa virusinfection in a human in need thereof by administering a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt or ester thereof.

In another embodiment, the method comprises treating a Junin virusinfection in a human in need thereof by administering a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt or ester thereof.

In another embodiment, the method of treating an Arenaviridae infectionin a human in need thereof comprises administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an effectiveamount of a Formula I compound, or a pharmaceutically acceptable salt orester thereof, in combination with a pharmaceutically acceptable diluentor carrier.

In another embodiment, the method of treating an Arenaviridae infectionin a human in need thereof comprises administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an effectiveamount of a Formula I compound, or a pharmaceutically acceptable salt orester thereof, in combination with at least one additional therapeuticagent.

In another embodiment, the method comprises administering atherapeutically effective amount of a combination pharmaceutical agentcomprising:

a) a first pharmaceutical composition comprising a compound of FormulaI; or a pharmaceutically acceptable salt, solvate, or ester thereof; and

b) a second pharmaceutical composition comprising at least oneadditional therapeutic agent active against infectious Arenaviridaeviruses.

In another embodiment, the present application provides for a method ofinhibiting an Arenaviridae RNA-dependent RNA polymerase, comprisingcontacting a cell infected with an Arenaviridae virus with an effectiveamount of a compound of Formula I; or a pharmaceutically acceptablesalts, solvate, and/or ester thereof.

In another embodiment, provided is the use of a compound of Formula I ora pharmaceutically acceptable salt, solvate, and/or ester thereof totreat a viral infection caused by an Arenaviridae virus.

Provided is a method for treating a Coronaviridae infection in a humanin need thereof comprising administering a therapeutically effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein:    -   each R¹ is H or halogen;    -   each R², R³, R⁴ or R⁵ is independently H, OR^(a), N(R^(a))₂, N₃,        CN, NO₂, S(O)_(n)R^(a), halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or        (C₂-C₈)substituted alkynyl;        -   or any two R², R³, R⁴ or R⁵ on adjacent carbon atoms when            taken together are —O(CO)O— or when taken together with the            ring carbon atoms to which they are attached form a double            bond;    -   R⁶ is OR^(a), N(R^(a))₂, N₃, CN, NO₂, S(O)_(n)R^(a), —C(═O)R¹¹,        —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹, —S(O)R¹¹, —S(O)₂R¹¹,        —S(O)(OR¹¹), —S(O)₂(OR¹¹), —SO₂NR¹¹R¹², halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl,        (C₂-C₈)substituted alkynyl, or C₆-C₂₀)aryl(C₁-C₈)alkyl;    -   R⁷ is selected from a group consisting of        -   a) H, —C(═O)R¹¹, —C(═O)_(0R) ¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹,            —S(O)R¹¹, —S(O)₂R¹¹, —S(O)(OR¹¹), —S(O)₂(OR¹¹), or            SO₂NR¹¹R¹²,            -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl,                (C₂-C₈)alkynyl or C₆-C₂₀)aryl(C₁-C₈)alkyl of each R¹¹ or                R¹² is, independently, optionally substituted with one                or more halo, hydroxy, CN, N₃, N(R^(a))₂ or OR^(a); and                wherein one or more of the non-terminal carbon atoms of                each said (C₁-C₈)alkyl may be optionally replaced with                —O—, —S— or —NR^(a)—,

-   -   -   -   wherein:                -   R^(c) is selected from phenyl, 1-naphthyl,                    2-naphthyl,

-   -   -   -   -   R^(d) is H or CH₃;                -   R^(e1) and R^(e2) are each independently H,                    (C₁-C₆)alkyl or benzyl;                -   R^(f) is selected from H, (C₁-C₈)alkyl, benzyl,                    (C₃-C₆)cycloalkyl, and —CH₂—(C₃-C₆)cycloalkyl;                -   R^(g) is selected from (C₁-C₈)alkyl,                    —O—(C₁-C₈)alkyl, benzyl, —O-benzyl,                    —CH₂(C₃-C₆)cycloalkyl, —O—CH₂(C₃-C₆)cycloalkyl, and                    CF₃; and                -   n′ is selected from 1, 2, 3, and 4; and

        -   d) a group of the formula:

-   -   -   -   wherein:                -   Q is O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;                -   Z¹ and Z², when taken together, are                    —Q¹(C(R^(y))₂)₃Q¹-;                -   wherein                -    each Q¹ is independently O, S, or NR; and                -    each R^(y) is independently H, F, Cl, Br, I, OH, R,                    —C(═Q²)R, —C(═Q²)OR, —C(═Q²)N(R)₂, —N(R)₂, —⁺N(R)₃,                    —SR, —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR),                    —OC(═Q¹)R, —OC(═Q²)OR, —OC(═Q²)(N(R)₂), —SC(═Q²)R,                    —SC(═Q²)OR, —SC(═Q²)(N(R)₂), —N(R)C(═Q²)R,                    —N(R)C(═Q²)OR, —N(R)C(═Q²)N(R)₂, —SO₂NR₂, —CN, —N₃,                    —NO₂, —OR, or Z³; or when taken together, two R^(y)                    on the same carbon atom form a carbocyclic ring of 3                    to 7 carbon atoms;                -    each Q² is independently, O, S, NR, ⁺N(O)(R),                    N(OR), ⁺N(O)(OR), or N—NR₂; or                -   Z¹ and Z² are each, independently, a group of the                    Formula Ia:

-   -   -   -   -   wherein:                -    each Q³ is independently a bond, O, CR₂, NR,                    ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S, S—S, S(O), or                    S(O)₂;                -    M2 is 0, 1 or 2;                -    each R^(x) is independently R^(y) or the formula:

-   -   -   -   -    wherein:                -    each M1a, M1c, and M1d is independently 0 or 1;                -    M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;                -    Z³ is Z⁴ or Z⁵;                -    Z⁴ is R, —C(Q²)R^(y), —C(Q²)Z⁵, —SO₂R^(y), or                    —SO₂Z⁵; and                -    Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is                    independently substituted with 0 to 3 R^(y) groups;

    -   R⁸ is halogen, NR¹¹R¹², N(R¹¹)OR¹¹, NR¹¹NR¹¹R¹², N₃, NO, NO₂,        CHO, CN, —CH(═NR¹¹), —CH═NNHR¹¹, —CH═N(OR¹¹), —CH(OR¹¹)₂,        —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹², —C(═O)OR¹¹, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl,        (C₆-C₂₀)aryl(C₁-C₈)alkyl, OR¹¹ or SR¹¹;

    -   each R⁹ or R¹⁰ is independently H, halogen, NR¹¹R¹², N(R¹¹)OR¹¹,        NR¹¹NR¹¹R¹², N₃, NO, NO₂, CHO, CN, —CH(═NR¹¹), —CH═NHNR¹¹,        —CH═N(OR¹¹), —CH(OR¹¹)₂, —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹²,        —C(═O)OR¹¹, —C(═O)OR¹¹, R¹¹, OR¹¹ or SR¹¹;

    -   each R¹¹ or R¹² is independently H, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl; or R¹¹ and R¹² taken together with a        nitrogen to which they are both attached form a 3 to 7 membered        heterocyclic ring wherein any one carbon atom of said        heterocyclic ring can optionally be replaced with —O—, —S—or        —NR^(a)—;

    -   each R^(a) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, —C(═O)R, —C(═O)OR, —C(═O)NR₂, —C(═O)SR,        —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), or —SO₂NR₂; wherein

    -   each R is independently H, (C₁-C₈) alkyl, (C₁-C₈) substituted        alkyl, (C₂-C₈)alkenyl, (C₂-C₈) substituted alkenyl, (C₂-C₈)        alkynyl, (C₂-C₈) substituted alkynyl, (C₆-C₂₀)aryl,        (C₆-C₂₀)substituted aryl, (C₂-C₂₀)heterocyclyl,        (C₂-C₂₀)substituted heterocyclyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl or        substituted (C₆-C₂₀)aryl(C₁-C₈)alkyl;

    -   each n is independently 0, 1, or 2; and

    -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl of each R², R³, R⁵, R⁶, R¹¹ or R¹² is,        independently, optionally substituted with one or more halo,        hydroxy, CN, N₃, N(R^(a))₂ or OR^(a); and wherein one or more of        the non-terminal carbon atoms of each said (C₁-C₈)alkyl may be        optionally replaced with —O—, —S—or NR^(a)—.

    -   In another embodiment, the method comprises administering a        therapeutically effective amount of a racemate, enantiomer,        diastereomer, tautomer, polymorph, pseudopolymorph, amorphous        form, hydrate or solvate of a compound of Formula I or a        pharmaceutically acceptable salt or ester thereof to a mammal in        need thereof.

    -   In another embodiment, the method comprises treating a        Coronaviridae infection in a human in need thereof by        administering a therapeutically effective amount of a compound        of Formula I or a pharmaceutically acceptable salt or ester        thereof.

In another embodiment, the method comprises treating a MERS virusinfection in a human in need thereof by administering a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt or ester thereof.

In another embodiment, the method comprises treating a SARS virusinfection in a human in need thereof by administering a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt or ester thereof.

In another embodiment, the method of treating a Coronaviridae infectionin a human in need thereof comprises administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an effectiveamount of a Formula I compound, or a pharmaceutically acceptable salt orester thereof, in combination with a pharmaceutically acceptable diluentor carrier.

In another embodiment, the method of treating a Coronaviridae infectionin a human in need thereof comprises administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an effectiveamount of a Formula I compound, or a pharmaceutically acceptable salt orester thereof, in combination with at least one additional therapeuticagent.

In another embodiment, the method comprises administering atherapeutically effective amount of a combination pharmaceutical agentcomprising:

a) a first pharmaceutical composition comprising a compound of FormulaI; or a pharmaceutically acceptable salt, solvate, or ester thereof; and

b) a second pharmaceutical composition comprising at least oneadditional therapeutic agent active against infectious Coronaviridaeviruses.

In another embodiment, the present application provides for a method ofinhibiting a Coronaviridae RNA-dependent RNA polymerase, comprisingcontacting a cell infected with a Coronaviridae virus with an effectiveamount of a compound of Formula I; or a pharmaceutically acceptablesalts, solvate, and/or ester thereof.

In another embodiment, provided is the use of a compound of Formula I ora pharmaceutically acceptable salt, solvate, and/or ester thereof totreat a viral infection caused by a Coronaviridae virus.

DESCRIPTION OF THE FIGURES

FIG. 1: Changes in body weight post infection in vehicle and Compound32-treated mice

FIG. 2A and FIG. 2B: Viral load in lung tissue at Day 2 and 5 postinfection in vehicle and Compound 32-treated mice

FIG. 3A-F: Whole Body Plethysmography of Mice Infected with SARS-CoV

FIG. 4A. Changes in body weight post infection in vehicle and Compound32-treated monkey

FIG. 4B. Changes in body temperature post infection in vehicle andCompound 32-treated monkey

FIG. 4C. Changes in respiratory rate post infection in vehicle andCompound 32-treated monkey

FIG. 5. Tissue viral RNA concentrations by treatment group. Viral loadwas measured qRT-PCR.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

When trade names are used herein, applicants intend to independentlyinclude the trade name product and the active pharmaceuticalingredient(s) of the trade name product.

As used herein, “a compound of the invention” or “a compound of FormulaI” means a compound of Formula I or a pharmaceutically acceptable salt,thereof. Similarly, with respect to isolatable intermediates, the phrase“a compound of Formula (number)” means a compound of that formula andpharmaceutically acceptable salts, thereof.

“Alkyl” is hydrocarbon containing normal, secondary, tertiary or cycliccarbon atoms. For example, an alkyl group can have 1 to 20 carbon atoms(i.e, C₁-C₂₀ alkyl), 1 to 8 carbon atoms (i.e., C₁-C₈ alkyl), or 1 to 6carbon atoms (i.e., C₁-C₆ alkyl). Examples of suitable alkyl groupsinclude, but are not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃),1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl,—CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl(i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, and octyl (—(CH₂)₇CH₃).

“Alkoxy” means a group having the formula —O-alkyl, in which an alkylgroup, as defined above, is attached to the parent molecule via anoxygen atom. The alkyl portion of an alkoxy group can have 1 to 20carbon atoms (i.e., C₁-C₂₀ alkoxy), 1 to 12 carbon atoms (i.e., C₁-C₁₂alkoxy), or 1 to 6 carbon atoms(i.e., C₁-C₆ alkoxy). Examples ofsuitable alkoxy groups include, but are not limited to, methoxy (—O—CH₃or —OMe), ethoxy (—OCH₂CH₃ or —OEt), t-butoxy (—O—C(CH₃)₃ or —OtBu) andthe like.

“Haloalkyl” is an alkyl group, as defined above, in which one or morehydrogen atoms of the alkyl group is replaced with a halogen atom. Thealkyl portion of a haloalkyl group can have 1 to 20 carbon atoms (i.e.,C₁-C₂₀ haloalkyl), 1 to 12 carbon atoms(i.e., C₁-C₁₂ haloalkyl), or 1 to6 carbon atoms(i.e., C₁-C₆ alkyl). Examples of suitable haloalkyl groupsinclude, but are not limited to, —CF₃, —CHF₂, —CFH₂, —CH₂CF₃, and thelike.

“Alkenyl” is a hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. For example, an alkenyl group can have 2to 20 carbon atoms (i.e., C₂-C₂₀ alkenyl), 2 to 8 carbon atoms (i.e.,C₂-C₈ alkenyl), or 2 to 6 carbon atoms (i.e., C₂-C₆ alkenyl). Examplesof suitable alkenyl groups include, but are not limited to, ethylene orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl (—C₅H₇), and5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂).

“Alkynyl” is a hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. For example, an alkynyl group can have 2to 20 carbon atoms (i.e., C₂-C₂₀ alkynyl), 2 to 8 carbon atoms (i.e.,C₂-C₈ alkyne,), or 2 to 6 carbon atoms (i.e., C₂-C₆ alkynyl). Examplesof suitable alkynyl groups include, but are not limited to, acetylenic(—C≡CH), propargyl (—CH₂C≡CH), and the like.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical having two monovalent radical centers derived by theremoval of two hydrogen atoms from the same or two different carbonatoms of a parent alkane. For example, an alkylene group can have 1 to20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typicalalkylene radicals include, but are not limited to, methylene (—CH₂—),1,1-ethyl (—CH(CH₃)—), 1,2-ethyl (—CH₂CH₂—), 1,1-propyl (—CH(CH₂CH₃)—),1,2-propyl (—CH₂CH(CH₃)—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl(—CH₂CH₂CH₂CH₂—), and the like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical having two monovalent radical centers derivedby the removal of two hydrogen atoms from the same or two differentcarbon atoms of a parent alkene. For example, and alkenylene group canhave 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms.Typical alkenylene radicals include, but are not limited to,1,2-ethylene (—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical having two monovalent radical centers derivedby the removal of two hydrogen atoms from the same or two differentcarbon atoms of a parent alkyne. For example, an alkynylene group canhave 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms.Typical alkynylene radicals include, but are not limited to, acetylene(—C≡C—), propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Amino” refers generally to a nitrogen radical which can be considered aderivative of ammonia, having the formula —N(X)₂, where each “X” isindependently H, substituted or unsubstituted alkyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,etc. The hybridization of the nitrogen is approximately sp^(a).Nonlimiting types of amino include NH₂, —N(alkyl)₂, —NH(alkyl),—N(carbocyclyl)₂, —NH(carbocyclyl), —N(heterocyclyl)₂,—NH(heterocyclyl), —N(aryl)₂, —NH(aryl), —N(alkyl)(aryl),—N(alkyl)(heterocyclyl), —N(carbocyclyl)(heterocyclyl),—N(aryl)(heteroaryl), —N(alkyl)(heteroaryl), etc. The term “alkylamino”refers to an amino group substituted with at least one alkyl group.Nonlimiting examples of amino groups include NH₂, —NH(CH₃), —N(CH₃)₂,—NH(CH₂CH₃), —N(CH₂CH₃)₂, —NH(phenyl), —N(phenyl)₂, —NH(benzyl),—N(benzyl)₂, etc. Substituted alkylamino refers generally to alkylaminogroups, as defined above, in which at least one substituted alkyl, asdefined herein, is attached to the amino nitrogen atom. Non-limitingexamples of substituted alkylamino includes —NH(alkylene-C(O)—OH),—NH(alkylene-C(O)—O-alkyl), —N(alkylene-C(O)—OH)₂,—N(alkylene-C(O)—O-alkyl)₂, etc.

“Aryl” means an aromatic hydrocarbon radical derived by the removal ofone hydrogen atom from a single carbon atom of a parent aromatic ringsystem. For example, an aryl group can have 6 to 20 carbon atoms, 6 to14 carbon atoms, or 6 to 10 carbon atoms. Typical aryl groups include,but are not limited to, radicals derived from benzene (e.g., phenyl),substituted benzene, naphthalene, anthracene, biphenyl, and the like.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. The arylalkyl group can comprise7 to 20 carbon atoms, e.g., the alkyl moiety is 1 to 6 carbon atoms andthe aryl moiety is 6 to 14 carbon atoms.

“Arylalkenyl” refers to an acyclic alkenyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, but also an sp² carbon atom, is replaced with an arylradical. The aryl portion of the arylalkenyl can include, for example,any of the aryl groups disclosed herein, and the alkenyl portion of thearylalkenyl can include, for example, any of the alkenyl groupsdisclosed herein. The arylalkenyl group can comprise 8 to 20 carbonatoms, e.g., the alkenyl moiety is 2 to 6 carbon atoms and the arylmoiety is 6 to 14 carbon atoms.

“Arylalkynyl” refers to an acyclic alkynyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, but also an sp carbon atom, is replaced with an arylradical. The aryl portion of the arylalkynyl can include, for example,any of the aryl groups disclosed herein, and the alkynyl portion of thearylalkynyl can include, for example, any of the alkynyl groupsdisclosed herein. The arylalkynyl group can comprise 8 to 20 carbonatoms, e.g., the alkynyl moiety is 2 to 6 carbon atoms and the arylmoiety is 6 to 14 carbon atoms.

The term “substituted” in reference to alkyl, alkylene, aryl, arylalkyl,alkoxy, heterocyclyl, heteroaryl, carbocyclyl, etc., for example,“substituted alkyl”, “substituted alkylene”, “substituted aryl”,“substituted arylalkyl”, “substituted heterocyclyl”, and “substitutedcarbocyclyl” means alkyl, alkylene, aryl, arylalkyl, heterocyclyl,carbocyclyl respectively, in which one or more hydrogen atoms are eachindependently replaced with a non-hydrogen substituent. Typicalsubstituents include, but are not limited to, —X, —R^(b), —O⁻, ═O,—OR^(b), —SR^(b), —S⁻, —NR^(b) ₂, ═NR^(b), —CX₃, —CN, —OCN, —SCN,—N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NHC(═O)R^(b), —OC(═O)R^(b),—NHC(═O)NR^(b) ₂, —S(═O)₂—, —S(═O)₂OH, —S(═O)₂R^(b), —OS(═O)OR^(b),—S(═O)₂NR^(b) ₂, —S(═O)R^(b), —OP(═O)(OR^(b))₂, —P(═O)(OR^(b))₂,—P(═O)(O⁻)₂, —P(═O)(OH)₂, —P(O)(OR^(b))(O⁻), —C(═O)R^(b), —C(═O)X,—C(S)R^(b), —C(O)OR^(b), —C(O)O⁻, —C(S)OR^(b), —C(O)SR^(b), —C(S)SR^(b),—C(O)NR^(b) ₂, —C(S)NR^(b) ₂, —C(═NR^(b))NR^(b) ₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R^(b) isindependently H, alkyl, aryl, arylalkyl, a heterocycle, or a protectinggroup or prodrug moiety. Alkylene, alkenylene, and alkynylene groups mayalso be similarly substituted. Unless otherwise indicated, when the term“substituted” is used in conjunction with groups such as arylalkyl,which have two or more moieties capable of substitution, thesubstituents can be attached to the aryl moiety, the alkyl moiety, orboth.

A “prodrug” is defined in the pharmaceutical field as a biologicallyinactive derivative of a drug that upon administration to the human bodyis converted to the biologically active parent drug according to somechemical or enzymatic pathway.

One skilled in the art will recognize that substituents and othermoieties of the compounds of Formula I-IV should be selected in order toprovide a compound which is sufficiently stable to provide apharmaceutically useful compound which can be formulated into anacceptably stable pharmaceutical composition. Compounds of Formula I-IVwhich have such stability are contemplated as falling within the scopeof the present invention.

“Heteroalkyl” refers to an alkyl group where one or more carbon atomshave been replaced with a heteroatom, such as, O, N, or S. For example,if the carbon atom of the alkyl group which is attached to the parentmolecule is replaced with a heteroatom (e.g., O, N, or S) the resultingheteroalkyl groups are, respectively, an alkoxy group (e.g., —OCH₃,etc.), an amine (e.g., —NHCH₃, —N(CH₃)₂, etc.), or a thioalkyl group(e.g., —SCH₃). If a non-terminal carbon atom of the alkyl group which isnot attached to the parent molecule is replaced with a heteroatom (e.g.,O, N, or S) the resulting heteroalkyl groups are, respectively, an alkylether (e.g., —CH₂CH₂—O—CH₃, etc.), an alkyl amine (e.g., —CH₂NHCH₃,—CH₂N(CH₃)₂, etc.), or a thioalkyl ether (e.g., —CH₂—S—CH₃). If aterminal carbon atom of the alkyl group is replaced with a heteroatom(e.g., O, N, or S), the resulting heteroalkyl groups are, respectively,a hydroxyalkyl group (e.g., —CH₂CH₂—OH), an aminoalkyl group (e.g.,—CH₂NH₂), or an alkyl thiol group (e.g., —CH₂CH₂—SH). A heteroalkylgroup can have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms,or 1 to 6 carbon atoms. A C₁-C₆ heteroalkyl group means a heteroalkylgroup having 1 to 6 carbon atoms.

“Heterocycle” or “heterocyclyl” as used herein includes by way ofexample and not limitation those heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment ofthe invention “heterocycle” includes a “carbocycle” as defined herein,wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replacedwith a heteroatom (e.g. O, N, or S). The terms “heterocycle” or“heterocyclyl” includes saturated rings, partially unsaturated rings,and aromatic rings (i.e., heteroaromatic rings). Substitutedheterocyclyls include, for example, heterocyclic rings substituted withany of the substituents disclosed herein including carbonyl groups. Anon-limiting example of a carbonyl substituted heterocyclyl is:

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,P-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,isatinoyl, and bis-tetrahydrofuranyl:

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

“Heterocyclylalkyl” refers to an acyclic alkyl radical in which one ofthe hydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heterocyclyl radical (i.e., aheterocyclyl-alkylene- moiety). Typical heterocyclyl alkyl groupsinclude, but are not limited to heterocyclyl-CH₂—,2-(heterocyclyl)ethan-1-yl, and the like, wherein the “heterocyclyl”portion includes any of the heterocyclyl groups described above,including those described in Principles of Modern HeterocyclicChemistry. One skilled in the art will also understand that theheterocyclyl group can be attached to the alkyl portion of theheterocyclyl alkyl by means of a carbon-carbon bond or acarbon-heteroatom bond, with the proviso that the resulting group ischemically stable. The heterocyclyl alkyl group comprises 3 to 20 carbonatoms, e.g., the alkyl portion of the arylalkyl group is 1 to 6 carbonatoms and the heterocyclyl moiety is 2 to 14 carbon atoms. Examples ofheterocyclylalkyls include by way of example and not limitation5-membered sulfur, oxygen, and/or nitrogen containing heterocycles suchas thiazolylmethyl, 2-thiazolylethan-1-yl, imidazolylmethyl,oxazolylmethyl, thiadiazolylmethyl, etc., 6-membered sulfur, oxygen,and/or nitrogen containing heterocycles such as piperidinylmethyl,piperazinylmethyl, morpholinylmethyl, pyridinylmethyl, pyridizylmethyl,pyrimidylmethyl, pyrazinylmethyl, etc.

“Heterocyclylalkenyl” refers to an acyclic alkenyl radical in which oneof the hydrogen atoms bonded to a carbon atom, typically a terminal orsp³ carbon atom, but also a sp² carbon atom, is replaced with aheterocyclyl radical (i.e., a heterocyclyl-alkenylene- moiety). Theheterocyclyl portion of the heterocyclyl alkenyl group includes any ofthe heterocyclyl groups described herein, including those described inPrinciples of Modern Heterocyclic Chemistry, and the alkenyl portion ofthe heterocyclyl alkenyl group includes any of the alkenyl groupsdisclosed herein. One skilled in the art will also understand that theheterocyclyl group can be attached to the alkenyl portion of theheterocyclyl alkenyl by means of a carbon-carbon bond or acarbon-heteroatom bond, with the proviso that the resulting group ischemically stable. The heterocyclyl alkenyl group comprises 4 to 20carbon atoms, e.g., the alkenyl portion of the heterocyclyl alkenylgroup is 2 to 6 carbon atoms and the heterocyclyl moiety is 2 to 14carbon atoms.

“Heterocyclylalkynyl” refers to an acyclic alkynyl radical in which oneof the hydrogen atoms bonded to a carbon atom, typically a terminal orsp^(a) carbon atom, but also an sp carbon atom, is replaced with aheterocyclyl radical (i.e., a heterocyclyl-alkynylene- moiety). Theheterocyclyl portion of the heterocyclyl alkynyl group includes any ofthe heterocyclyl groups described herein, including those described inPrinciples of Modern Heterocyclic Chemistry, and the alkynyl portion ofthe heterocyclyl alkynyl group includes any of the alkynyl groupsdisclosed herein. One skilled in the art will also understand that theheterocyclyl group can be attached to the alkynyl portion of theheterocyclyl alkynyl by means of a carbon-carbon bond or acarbon-heteroatom bond, with the proviso that the resulting group ischemically stable. The heterocyclyl alkynyl group comprises 4 to 20carbon atoms, e.g., the alkynyl portion of the heterocyclyl alkynylgroup is 2 to 6 carbon atoms and the heterocyclyl moiety is 2 to 14carbon atoms.

“Heteroaryl” refers to an aromatic heterocyclyl having at least oneheteroatom in the ring. Non-limiting examples of suitable heteroatomswhich can be included in the aromatic ring include oxygen, sulfur, andnitrogen. Non-limiting examples of heteroaryl rings include all of thosearomatic rings listed in the definition of “heterocyclyl”, includingpyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl,thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl,thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl,pyridazyl, pyrimidyl, pyrazyl, etc.

“Carbocycle” or “carbocyclyl” refers to a saturated (i.e., cycloalkyl),partially unsaturated (e.g., cycloakenyl, cycloalkadienyl, etc.) oraromatic ring having 3 to 7 carbon atoms as a monocycle, 7 to 12 carbonatoms as a bicycle, and up to about 20 carbon atoms as a polycycle.Monocyclic carbocycles have 3 to 7 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.,arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo [5,6] or [6,6] system, or spiro-fusedrings. Non-limiting examples of monocyclic carbocycles includecyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and phenyl. Non-limiting examplesof bicyclo carbocycles includes naphthyl, tetrahydronapthalene, anddecaline.

“Carbocyclylalkyl” refers to an acyclic akyl radical in which one of thehydrogen atoms bonded to a carbon atom is replaced with a carbocyclylradical as described herein. Typical, but non-limiting, examples ofcarbocyclylalkyl groups include cyclopropylmethyl, cyclopropylethyl,cyclobutylmethyl, cyclopentylmethyl and cyclohexylmethyl.

“Arylheteroalkyl” refers to a heteroalkyl as defined herein, in which ahydrogen atom (which may be attached either to a carbon atom or aheteroatom) has been replaced with an aryl group as defined herein. Thearyl groups may be bonded to a carbon atom of the heteroalkyl group, orto a heteroatom of the heteroalkyl group, provided that the resultingarylheteroalkyl group provides a chemically stable moiety. For example,an arylheteroalkyl group can have the general formulae-alkylene-O-aryl,-alkylene-O-alkylene-aryl, -alkylene-NH-aryl,-alkylene-NH-alkylene-aryl, -alkylene—S—aryl, -alkylene—S—alkylene-aryl,etc. In addition, any of the alkylene moieties in the general formulaeabove can be further substituted with any of the substituents defined orexemplified herein.

“Heteroarylalkyl” refers to an alkyl group, as defined herein, in whicha hydrogen atom has been replaced with a heteroaryl group as definedherein. Non-limiting examples of heteroaryl alkyl include—CH₂-pyridinyl, —CH₂-pyrrolyl, —CH₂-oxazolyl, —CH₂-indolyl,—CH₂-isoindolyl, —CH₂-purinyl, —CH₂-furanyl, —CH₂-thienyl,—CH₂-benzofuranyl, —CH₂-benzothiophenyl, —CH₂-carbazolyl,—CH₂-imidazolyl, —CH₂-thiazolyl, —CH₂-isoxazolyl, —CH₂-pyrazolyl,—CH₂-isothiazolyl, —CH₂-quinolyl, —CH₂-isoquinolyl, —CH₂-pyridazyl,—CH₂-pyrimidyl, —CH₂-pyrazyl, —CH(CH₃)-pyridinyl, —CH(CH₃)-pyrrolyl,—CH(CH₃)-oxazolyl, —CH(CH₃)-indolyl, —CH(CH₃)-isoindolyl,—CH(CH₃)-purinyl, —CH(CH₃)-furanyl, —CH(CH₃)-thienyl,—CH(CH₃)-benzofuranyl, —CH(CH₃)-benzothiophenyl, —CH(CH₃)-carbazolyl,—CH(CH₃)-imidazolyl, —CH(CH₃)-thiazolyl, —CH(CH₃)-isoxazolyl,—CH(CH₃)-pyrazolyl, —CH(CH₃)-isothiazolyl, —CH(CH₃)-quinolyl,—CH(CH₃)-isoquinolyl, —CH(CH₃)-pyridazyl, —CH(CH₃)-pyrimidyl,—CH(CH₃)-pyrazyl, etc.

The term “optionally substituted” in reference to a particular moiety ofthe compound of Formula I-IV (e.g., an optionally substituted arylgroup) refers to a moiety wherein all substituents are hydrogen orwherein one or more of the hydrogens of the moiety may be replaced bysubstituents such as those listed under the definition of “substituted”.

The term “optionally replaced” in reference to a particular moiety ofthe compound of Formula I-IV (e.g., the carbon atoms of said(C₁-C₈)alkyl may be optionally replaced by —O—, —S—, or —NR^(a)—) meansthat one or more of the methylene groups of the (C₁-C₈)alkyl may bereplaced by 0, 1, 2, or more of the groups specified (e.g., —O—, —S—, or—NR^(a)—).

The term “non-terminal carbon atom(s)” in reference to an alkyl,alkenyl, alkynyl, alkylene, alkenylene, or alkynylene moiety refers tothe carbon atoms in the moiety that intervene between the first carbonatom of the moiety and the last carbon atom in the moiety. Therefore, byway of example and not limitation, in the alkyl moiety—CH₂(C*)H₂(C*)H₂CH₃ or alkylene moiety —CH₂(C*)H₂(C*)H₂CH₂— the C* atomswould be considered to be the non-terminal carbon atoms.

Certain Q and Q¹ alternatives are nitrogen oxides such as ⁺N(O)(R) or⁺N(O)(OR). These nitrogen oxides, as shown here attached to a carbonatom, can also be represented by charge separated groups such as

respectively, and are intended to be equivalent to the aforementionedrepresentations for the purposes of describing this invention.

“Linker” or “link” means a chemical moiety comprising a covalent bond ora chain of atoms. Linkers include repeating units of alkyloxy (e.g.polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g.polyethyleneamino, Jeffamine™); and diacid ester and amides includingsuccinate, succinamide, diglycolate, malonate, and caproamide. The termssuch as “oxygen-linked”, “nitrogen-linked”, “carbon-linked”,“sulfur-linked”, or “phosphorous-linked” mean that if a bond between twomoieties can be formed by using more than one type of atom in a moiety,then the bond formed between the moieties is through the atom specified.For example, a nitrogen-linked amino acid would be bonded through anitrogen atom of the amino acid rather than through an oxygen or carbonatom of the amino acid.

In some embodiments of the compounds of Formula I-IV, one or more of Z¹or Z² are independently a radical of a nitrogen-linked naturallyoccurring a-amino acid ester. Examples of naturally occurring aminoacids include isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, valine, alanine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine,serine, tyrosine, arginine, histidine, ornithine and taurine. The estersof these amino acids comprise any of those described for the substituentR, particularly those in which R is optionally substituted (C₁-C₈)alkyl.

The term “purine” or “pyrimidine” base comprises, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-allylaminopurine, N⁶-thioallyl purine, N²-alkylpurines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-5-iodopyrimidine, C⁶-iodo-pyrimidine, C⁵-Br-vinyl pyrimidine,C⁶-Br-vinyl pyriniidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloropurine.The purine and pyrimidine bases of Formula I-III are linked to theribose sugar, or analog thereof, through a nitrogen atom of the base.Functional oxygen and nitrogen groups on the base can be protected asnecessary or desired. Suitable protecting groups are well known to thoseskilled in the art, and include trimethylsilyl, dimethylhexylsilyl,t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups,and acyl groups such as acetyl and propionyl, methanesulfonyl, andp-toluenesulfonyl.

Unless otherwise specified, the carbon atoms of the compounds of FormulaI-IV are intended to have a valence of four. In some chemical structurerepresentations where carbon atoms do not have a sufficient number ofvariables attached to produce a valence of four, the remaining carbonsubstituents needed to provide a valence of four should be assumed to behydrogen. For example,

has the same meaning as

“Protecting group” refers to a moiety of a compound that masks or altersthe properties of a functional group or the properties of the compoundas a whole. The chemical substructure of a protecting group varieswidely. One function of a protecting group is to serve as anintermediate in the synthesis of the parental drug substance. Chemicalprotecting groups and strategies for protection/deprotection are wellknown in the art. See: “Protective Groups in Organic Chemistry”,Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991. Protectinggroups are often utilized to mask the reactivity of certain functionalgroups, to assist in the efficiency of desired chemical reactions, e.g.making and breaking chemical bonds in an ordered and planned fashion.Protection of functional groups of a compound alters other physicalproperties besides the reactivity of the protected functional group,such as the polarity, lipophilicity (hydrophobicity), and otherproperties which can be measured by common analytical tools. Chemicallyprotected intermediates may themselves be biologically active orinactive. “Hydroxy protecting groups” refers to those protecting groupsuseful for protecting hydroxy groups (—OH).

Protected compounds may also exhibit altered, and in some cases,optimized properties in vitro and in vivo, such as passage throughcellular membranes and resistance to enzymatic degradation orsequestration. In this role, protected compounds with intendedtherapeutic effects may be referred to as prodrugs. Another function ofa protecting group is to convert the parental drug into a prodrug,whereby the parental drug is released upon conversion of the prodrug invivo. Because active prodrugs may be absorbed more effectively than theparental drug, prodrugs may possess greater potency in vivo than theparental drug. Protecting groups are removed either in vitro, in theinstance of chemical intermediates, or in vivo, in the case of prodrugs.With chemical intermediates, it is not particularly important that theresulting products after deprotection, e.g. alcohols, be physiologicallyacceptable, although in general it is more desirable if the products arepharmacologically innocuous.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, reactivities and biologicalproperties. For example, the compounds of Formula I-IV may have a chiralphosphorus atom when R⁷ is

and Z¹ and Z² are different. When at least one of either Z¹ or Z² alsohas a chiral center, for example with Z¹ or Z² is a nitrogen-linked,chiral, naturally occurring a-amino acid ester, then the compound ofFormula I-IV will exists as diastereomers because there are two centersof chirality in the molecule. All such diastereomers and their usesdescribed herein are encompassed by the instant invention. Mixtures ofdiastereomers may be separate under high resolution analyticalprocedures such as electrophoresis, crystallization and/orchromatography. Diastereomers may have different physical attributessuch as, but not limited to, solubility, chemical stabilities andcrystallinity and may also have different biological properties such as,but not limited to, enzymatic stability, absorption and metabolicstability.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (e.g.,includes the degree of error associated with measurement of theparticular quantity).

The term “treating”, as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition to which such term applies, or one or moresymptoms of such disorder or condition. The term “treatment”, as usedherein, refers to the act of treating, as “treating” is definedimmediately above.

The term “therapeutically effective amount”, as used herein, is theamount of compound of Formula I-IV present in a composition describedherein that is needed to provide a desired level of drug in thesecretions and tissues of the airways and lungs, or alternatively, inthe bloodstream of a subject to be treated to give an anticipatedphysiological response or desired biological effect when such acomposition is administered by the chosen route of administration. Theprecise amount will depend upon numerous factors, for example theparticular compound of Formula I-IV, the specific activity of thecomposition, the delivery device employed, the physical characteristicsof the composition, its intended use, as well as patient considerationssuch as severity of the disease state, patient cooperation, etc., andcan readily be determined by one skilled in the art based upon theinformation provided herein.

The term “normal saline” means a water solution containing 0.9% (w/v)NaCl.

The term “hypertonic saline” means a water solution containing greaterthan 0.9% (w/v) NaCl. For example, 3% hypertonic saline would contain 3%(w/v) NaCl.

“Forming a reaction mixture” refers to the process of bringing intocontact at least two distinct species such that they mix together andcan react. It should be appreciated, however, the resulting reactionproduct can be produced directly from a reaction between the addedreagents or from an intermediate from one or more of the added reagentswhich can be produced in the reaction mixture.

“Coupling agent” refers to an agent capable of coupling two disparatecompounds. Coupling agents can be catalytic or stoichiometric. Forexample, the coupling agents can be a lithium based coupling agent or amagnesium based coupling agent such as a Grignard reagent. Exemplarycoupling agents include, but are not limited to, n-BuLi, MgCl₂, iPrMgCl,tBuMgCl, PhMgCl or combinations thereof.

“Silane” refers to a silicon containing group having the formula SiR4,where each R group can be alkyl, alkenyl, cycloalkyl, phenyl, or othersilicon containing groups. When the silane is linked to anothercompound, the silane is referred to as a “silyl” and has the formula—SiR₃.

“Halo-silane” refers to a silane having at least one halogen grouplinked to the silicon atom. Representative halo-silanes have the formulaHalo-SiR₃, where each R group can be alkyl, alkenyl, cycloalkyl, phenyl,or other silicon containing groups. Specific halo-silanes includeCl—Si(CH₃)₃, and Cl—Si(CH₃)₂CH₂CH₂Si(CH₃)₂—Cl.

“Non-nucleophilic base” refers to an electron donor, a Lewis base, suchas nitrogen bases including triethylamine, diisopropylethyl amine,N,N-diethylaniline, pyridine, 2,6-lutidine, 2,4,6-collidine,4-dimethylaminopyridine, and quinuclidine.

“Leaving group” refers to groups that maintain the bonding electron pairduring heterolytic bond cleavage. For example, a leaving group isreadily displaced during a nucleophilic displacement reaction. Suitableleaving groups include, but are not limited to, chloride, bromide,mesylate, tosylate, triflate, 4-nitrobenzenesulfonate,4-chlorobenzenesulfonate, 4-nitrophenoxy, pentafluorophenoxy, etc. Oneof skill in the art will recognize other leaving groups useful in thepresent invention.

“Deprotection agent” refers to any agent capable of removing aprotecting group. The deprotection agent will depend on the type ofprotecting group used. Representative deprotection agents are known inthe art and can be found in Protective Groups in Organic Chemistry,Peter G. M. Wuts and Theodora W. Greene, 4th Ed., 2006.

II. COMPOUNDS OF THE PRESENT INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingdescription, structures and formulas. While the invention will bedescribed in conjunction with the enumerated embodiments, it will beunderstood that they are not intended to limit the invention to thoseembodiments. On the contrary, the invention is intended to cover allalternatives, modifications, and equivalents, which may be includedwithin the scope of the present invention.

Provided is a method for treating an Arenaviridae infection in a humanin need thereof comprising administering a therapeutically effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein:    -   each R¹ is H or halogen;    -   each R², R³, R⁴ or R⁵ is independently H, OR^(a), N(R^(a))₂, N₃,        CN, NO₂, S(O)_(n)R^(a), halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or        (C₂-C₈)substituted alkynyl;        -   or any two R², R³, R⁴ or R⁵ on adjacent carbon atoms when            taken together are —O(CO)O— or when taken together with the            ring carbon atoms to which they are attached form a double            bond;    -   R⁶ is OR^(a), N(R^(a))₂, N₃, CN, NO₂, S(O)_(n)R^(a), —C(═O)R¹¹,        —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹, —S(O)R¹¹, —S(O)₂R¹¹,        —S(O)(OR¹¹), —S(O)₂(OR¹¹), —SO₂NR¹¹R¹², halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl,        (C₂-C₈)substituted alkynyl, or (C₆-C₂₀)aryl(C₁-C₈)alkyl;    -   R⁷ is selected from a group consisting of        -   a) H, —C(═O)R¹¹, —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹,            —S(O)R¹¹, —S(O)₂R¹¹, —S(O)(OR¹¹), —S(O)₂(OR¹¹), or            —SO₂NR¹¹R¹²,            -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl,                (C₂-C₈)alkynyl or (C₆-C₂₀)aryl(C₁-C₈)alkyl of each R¹¹                or R¹² is, independently, optionally substituted with                one or more halo, hydroxy, CN, N₃, N(R^(a))₂ or OR^(a);                and wherein one or more of the non-terminal carbon atoms                of each said (C₁-C₈)alkyl may be optionally replaced                with —O—, —S— or —NR^(a)—,

-   -   -   -   wherein:                -   R^(c) is selected from phenyl, 1-naphthyl,                    2-naphthyl,

-   -   -   -   -   R^(d) is H or CH₃;                -   R^(e1) and R^(e2) are each independently H,                    (C₁-C₆)alkyl or benzyl;                -   R^(f) is selected from H, (C₁-C₈)alkyl, benzyl,                    (C₃-C₆)cycloalkyl, and —CH₂(C₃-C₆)cycloalkyl;                -   R^(g) is selected from (C₁-C₈)alkyl,                    —O—(C₁-C₈)alkyl, benzyl, —O—benzyl,                    —CH₂—(C₃-C₆)cycloalkyl, —O—CH₂—(C₃-C₆)cycloalkyl,                    and CF₃; and                -   n′ is selected from 1, 2, 3, and 4; and

        -   d) a group of the formula:

-   -   -   -   wherein:                -   Q is O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;                -   Z¹ and Z², when taken together, are                    —Q¹(C(R^(Y))₂)₃Q¹-;                -   wherein                -    each Q¹ is independently O, S, or NR; and                -    each R^(y) is independently H, F, Cl, Br, I, OH, R,                    —C(═Q²)R, —C(═Q²)OR, —C(═Q²)N(R)₂, —N(R)₂, —⁺N(R)₃,                    —SR, —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR),                    —OC(═Q¹)R, —OC(═Q²)OR, —OC(═Q²)(N(R)₂), —SC(═Q²)R,                    —SC(═Q²)OR, —SC(═Q²)(N(R)₂), —N(R)C(═Q²)R,                    —N(R)C(═Q²)OR, —N(R)C(═Q²)N(R)₂, —SO₂NR₂, —CN, —N₃,                    —NO₂, —OR, or Z³; or when taken together, two R^(y)                    on the same carbon atom form a carbocyclic ring of 3                    to 7 carbon atoms;                -    each Q² is independently, O, S, NR, ⁺N(O)(R),                    N(OR), ⁺N(O)(OR), or N—NR₂; or                -   Z¹ and Z² are each, independently, a group of the                    Formula Ia:

-   -   -   -   -   wherein:                -    each Q³ is independently a bond, O, CR₂, NR,                    ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S, S—S, S(O), or                    S(O)₂;                -    M2 is 0, 1 or 2;                -    each R^(x) is independently R^(y) or the formula:

-   -   -   -   -    wherein:                -    each M1a, M1c, and M1d is independently 0 or 1;                -    M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;                -    Z³ is Z⁴ or Z⁵;                -    Z⁴ is R, —C(Q²)R^(y), —C(Q²)Z⁵, —SO₂R^(y), or                    —SO₂Z⁵; and                -    Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is                    independently substituted with 0 to 3 R^(y) groups;

    -   R⁸ is halogen, NR¹¹R¹², N(R¹¹)OR¹¹, NR¹¹NR¹¹R¹², N₃, NO, NO₂,        CHO, CN, —CH(═NR¹¹), —CH═NNHR¹¹, —CH═N(OR¹¹), —CH(OR¹¹)₂,        —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹², —C(═O)OR¹¹, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl,        (C₆-C₂₀)aryl(C₁-C₈)alkyl, OR¹¹ or SR¹¹;

    -   each R⁹ or R¹⁰ is independently H, halogen, NR¹¹R¹²,        NR¹¹NR¹¹R¹², N₃, NO, NO₂, CHO, CN, —CH(═NR¹¹), —CH═NHNR¹¹,        —CH═N(OR¹¹), —CH(OR¹¹)₂, —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹²,        —C(═O)OR¹¹, R¹¹, OR¹¹ or SR¹¹;

    -   each R¹¹ or R¹² is independently H, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl; or R¹¹ and R¹² taken together with a        nitrogen to which they are both attached form a 3 to 7 membered        heterocyclic ring wherein any one carbon atom of said        heterocyclic ring can optionally be replaced with —O—, —S—or        —NR^(a)—;

    -   each R^(a) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, —C(═O)R, —C(═O)OR, —C(═O)NR₂, —C(═O)SR,        —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), or —SO₂NR₂; wherein

    -   each R is independently H, (C₁-C₈) alkyl, (C₁-C₈) substituted        alkyl, (C₂-C₈)alkenyl, (C₂-C₈) substituted alkenyl, (C₂-C₈)        alkynyl, (C₂-C₈) substituted alkynyl, (C₆-C₂₀)aryl,        (C₆-C₂₀)substituted aryl, (C₂-C₂₀)heterocyclyl,        (C₂-C₂₀)substituted heterocyclyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl or        substituted (C₆-C₂₀)aryl(C₁-C₈)alkyl;

    -   each n is independently 0, 1, or 2; and

    -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or        C₆-C₂₀)aryl(C₁-C₈)alkyl of each R², R³, R⁵, R⁶, R¹¹ or R¹² is,        independently, optionally substituted with one or more halo,        hydroxy, CN, N₃, N(R^(a))₂ or OR^(a); and wherein one or more of        the non-terminal carbon atoms of each said (C₁-C₈)alkyl may be        optionally replaced with —O—, —S—or —NR^(a)—.

In another embodiment, provided is a method of treating an Arenaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I representedby Formula II:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein    -   R¹, R³, R⁵, R⁷, R⁸ and R⁹ are as defined above for Formula I;    -   each R² is OR^(a) or halogen; and    -   R⁶ is OR^(a), N(R^(a))₂, N₃, CN, S(O)_(n)R^(a), —C(═O)R¹¹,        —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹, —S(O)R¹¹, —S(O)₂R¹¹,        —S(O)(OR¹¹), —S(O)₂(OR¹¹), SO₂NR¹¹R¹², halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or        (C₂-C₈)substituted alkynyl.

In one embodiment of the method of treating an Arenaviridae infection byadministering a compound of Formula II, R¹ of Formula II is H. Inanother aspect of this embodiment R⁶ of Formula II is N₃, CN, halogen,(C₁-C₈)alkyl, (C₁-C₈)substituted alkyl, (C₂-C₈)alkenyl,(C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or (C₂-C₈)substitutedalkynyl. In another aspect of this embodiment, R⁶ of Formula II is CN,methyl, ethenyl, or ethynyl. In another aspect of this embodiment, R⁶ ofFormula II is CN. In another aspect of this embodiment, R⁶ of Formula IIis methyl. In another aspect of this embodiment, R⁵ of Formula II is H.In another aspect of this embodiment, R² of Formula II is OR^(a). Inanother aspect of this embodiment, R² of Formula II is OH. In anotheraspect of this embodiment, R² of Formula II is F. In another aspect ofthis embodiment, R³ of Formula II is OR^(a). In another aspect of thisembodiment, R³ of Formula II is OH, —OC(═O)R¹¹, or —OC(═O)OR¹¹. Inanother aspect of this embodiment, R³ of Formula II is OH. In anotheraspect of this embodiment, R⁸ of Formula II is NR¹¹R¹². In anotheraspect of this embodiment, R⁸ of Formula II is NH₂. In another aspect ofthis embodiment, R⁸ of Formula II is OR¹¹. In another aspect of thisembodiment, R⁸ of Formula II is OH. In another aspect of thisembodiment, R⁹ of Formula II is H. In another aspect of this embodiment,R⁹ of Formula II is NR¹¹R¹². In another aspect of this embodiment, R⁹ ofFormula II is NH₂. In another aspect of this embodiment, R⁷ of FormulaII is H, —C(═O)R¹¹, —C(═O)OR¹¹ or

In another aspect of this embodiment, R⁷ of Formula II is H. In anotheraspect of this embodiment, R⁷ of Formula II is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula II, theArenaviridae infection is caused by an Arenaviridae virus. In anotheraspect of this embodiment, the Arenaviridae virus is a Lassa virus orJunin virus. In another aspect of this embodiment, the Arenaviridaevirus is a Lassa virus. In another aspect of this embodiment, theArenaviridae virus is a Junin virus. In another aspect of thisembodiment, the Arenaviridae virus is caused by a Lassa virus caused bya strain selected from Josiah, NL, z148, Macenta, AV, and CSF.

In another aspect of this embodiment, the Arenaviridae infection iscaused by Allpahuayo virus (ALLY), Amapari virus (AMAV), Bear Canyonvirus (BCNV), Catarina virus, Chapare virus, Cupixi virus (CPXV),Dandenong virus, Flexal virus (FLEV), Guanarito virus (GTOV), Ippy virus(IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV), Latinovirus (LATV), Lymphocytic choriomeningitis virus (LCMV), Lujo virus,Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus(MOPV), Oliveros virus (OLVV), Parana virus (PARV), Pichinde virus(PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), SkinnerTank virus, Tacaribe virus (TCRV), Tamiami virus (TAMV), or WhitewaterArroyo virus (WWAV).

In another embodiment, provided is a method of treating an Arenaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I representedby Formula III:

or a pharmaceutically acceptable salt or ester, thereof;

-   -   wherein    -   R⁶, R⁷, R⁸ and R⁹ are as defined above for Formula II;    -   each R² is OR^(a) or F; and    -   each R³ is OR^(a).

In one embodiment of the method of treating an Arenaviridae infectioncomprising administering a compound of Formula III, R⁶ of Formula III isN₃, CN, halogen, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl, (C₂-C₈)alkenyl,(C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or (C₂-C₈)substitutedalkynyl. In another aspect of this embodiment, R⁶ of Formula III is CN,methyl, ethenyl, or ethynyl. In another aspect of this embodiment, R⁶ ofFormula III is CN. In another aspect of this embodiment, R⁶ of FormulaIII is methyl. In another aspect of this embodiment, R² of Formula IIIis OR^(a). In another aspect of this embodiment, R² of Formula III isOH. In another aspect of this embodiment, R² of Formula III is F. Inanother aspect of this embodiment, R³ of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R³ of Formula IIIis OH. In another aspect of this embodiment, R⁸ of Formula III isNR¹¹R¹². In another aspect of this embodiment, R⁸ of Formula III is NH₂.In another aspect of this embodiment, R⁸ of Formula III is OR¹¹. Inanother aspect of this embodiment, R⁸ of Formula III is OH. In anotheraspect of this embodiment, R⁹ of Formula III is H. In another aspect ofthis embodiment, R⁹ of Formula III is NR¹¹R¹². In another aspect of thisembodiment, R⁹ of Formula III is NH₂. In another aspect of thisembodiment, R⁷ of Formula III is H, —C(═O)R¹¹, —C(═O)OR¹¹ or

In another aspect of this embodiment, R⁷ of Formula III is H. In anotheraspect of this embodiment, R⁷ of Formula III is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula III, R⁶ ofFormula III is N₃, CN, halogen, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl,(C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or(C₂-C₈)substituted alkynyl and R⁸ is NH₂. In another aspect of thisembodiment, R⁶ of Formula III is CN, methyl, ethenyl, or ethynyl. Inanother aspect of this embodiment, R⁶ of Formula III is CN. In anotheraspect of this embodiment, R⁶ of Formula III is methyl. In anotheraspect of this embodiment, R² of Formula III is OR^(a). In anotheraspect of this embodiment, R² of Formula III is OH, —OC(═O)R¹¹, or—OC(═O)OR¹¹. In another aspect of this embodiment, R² of Formula III isOH. In another aspect of this embodiment, R² of Formula III is F. Inanother aspect of this embodiment, R³ of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R³ of Formula IIIis OH. In another aspect of this embodiment, R⁹ of Formula III is H. Inanother aspect of this embodiment, R⁹ of Formula III is NR¹¹R¹². Inanother aspect of this embodiment, R⁹ of Formula III is NH₂. In anotheraspect of this embodiment, R⁷ of Formula III is H, —C(═O)R¹¹, —C(═O)OR¹¹or

-   In another aspect of this embodiment, R⁷ of Formula III is H. In    another aspect of this embodiment, R⁷ of Formula III is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula III, R⁶ ofFormula III is CN, methyl, ethenyl, or ethynyl, R⁸ is NH₂, and R⁹ is H.In another aspect of this embodiment, R⁶ of Formula III is CN. Inanother aspect of this embodiment, R⁶ of Formula III is methyl. Inanother aspect of this embodiment, R² of Formula III is OR^(a). Inanother aspect of this embodiment, R² of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R² of Formula IIIis OH. In another aspect of this embodiment, R² of Formula III is F. Inanother aspect of this embodiment, R³ of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R³ of Formula IIIis OH. In another aspect of this embodiment, R⁷ of Formula III is H,—C(═O)R¹¹, —C(═O)OR¹¹ or

In another aspect of this embodiment, R⁷ of Formula III is H. In anotheraspect of this embodiment, R⁷ of Formula III is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula III, theArenaviridae infection is caused by an Arenaviridae virus. In anotheraspect of this embodiment, the Arenaviridae virus is a Lassa virus orJunin virus. In another aspect of this embodiment, the Arenaviridaevirus is a Lassa virus. In another aspect of this embodiment, theArenaviridae virus is a Junin virus. In another aspect of thisembodiment, the Arenaviridae virus is caused by a Lassa virus caused bya strain selected from Josiah, NL, z148, Macenta, AV, and CSF.

In another aspect of this embodiment, the Arenaviridae infection iscaused by Allpahuayo virus (ALLY), Amapari virus (AMAV), Bear Canyonvirus (BCNV), Catarina virus, Chapare virus, Cupixi virus (CPXV),Dandenong virus, Flexal virus (FLEV), Guanarito virus (GTOV), Ippy virus(IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV), Latinovirus (LATV), Lymphocytic choriomeningitis virus (LCMV), Lujo virus,Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus(MOPV), Oliveros virus (OLVV), Parana virus (PARV), Pichinde virus(PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), SkinnerTank virus, Tacaribe virus (TCRV), Tamiami virus (TAMV), or WhitewaterArroyo virus (WWAV).

In another embodiment, provided is a method of treating an Arenaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I representedby Formula IV:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein R⁷ is as defined above for Formula I.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ can beH. In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ isselected from the group of a), b), or c) as defined for Formula I.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein Z¹ and Z² are each, independently, a group having the structure:

and Z³ is Z⁵.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein Z¹ and Z² are each, independently, a group having the structure:

and Z³ is Z⁵.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein each Q^(3b) is, independently, O or N(R). In another embodiment,each Q^(3b) is O and each R^(x) is independently:

wherein M12c is 1, 2 or 3 and each Q³ is independently a bond, O, CR₂,or S.

In some embodiments, R^(e1) and R^(e2) can each independently be H,C₁-C₆ alkyl or benzyl. In some embodiments, R^(e1) can be H, C₁-C₆ alkylor benzyl, and R^(e2) can be H or C₁-C₆ alkyl. In some embodiments,R^(e1) and R^(e2) can each independently be H or C₁-C₆ alkyl. In someembodiments, R^(e1) and R^(e2) can each independently be H or benzyl. Insome embodiments, R^(e1) can be H, methyl or benzyl, and R^(e2) can be Hor methyl. In some embodiments, R^(e1) can be H or methyl, and R^(e2)can be H or methyl. In some embodiments, R^(e1) can be methyl, andR^(e2) can be H or methyl. In some embodiments, R^(e1) can be H orbenzyl, and R^(e2) can be H or methyl.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein R^(f) is selected from the group of from H, C₁-C₈ alkyl, benzyl,C₃-C₆ cycloalkyl, and —CH₂—C₃-C₆ cycloalkyl. In another embodiment of acompound of Formula IV, R^(f) is C₁-C₈ alkyl. In another embodiment of acompound of Formula IV, R^(f) is 2-ethylbutyl.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein

-   -   R^(f) is selected from H, C₁-C₈ alkyl, benzyl, C₃-C₆ cycloalkyl,        and CH₂—C₃-C₆ cycloalkyl; and    -   R^(g) is selected from C₁-C₈ alkyl, —O—C₁-C₈ alkyl, benzyl,        —O-benzyl, —CH₂-C₃-C₆ cycloalkyl, —O—CH₂—C₃-C₆ cycloalkyl, and        CF₃.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein R^(f) is selected from H, C₁-C₈ alkyl, benzyl, C₃-C₆ cycloalkyl,and —CH₂—C₃-C₆ cycloalkyl. In another embodiment of a compound ofFormula IV, R^(f)is C₁-C₈ alkyl. In another embodiment of a compound ofFormula IV, R^(f) is C₁-C₆ alkyl. In another embodiment of a compound ofFormula IV, R^(f) is 2-ethylbutyl.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is:

wherein R^(g) is selected from C₁-C₈ alkyl, —O—C₁-C₈ alkyl, benzyl,—O-benzyl, —CH₂-C₃-C₆ cycloalkyl, —O—CH₂-C₃-C₆ cycloalkyl, and CF₃. Inanother embodiment of a compound of Formula IV, R^(f) is C₁-C₈ alkyl. Inanother embodiment of a compound of Formula IV, R^(f) is C₁-C₆ alkyl.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ isselected from the group of:

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, Z¹ and Z²can each be:

In another embodiment, provided is a method of treating an Arenaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formulas I-IV, whereinR¹¹ or R¹² is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl, optionally substituted aryl,optionally substituted heteroaryl, —C(═O)(C₁-C₈)alkyl,—S(O)_(n)(C₁-C₈)alkyl or aryl(C₁-C₈)alkyl. In another embodiment, R¹¹and R¹² taken together with a nitrogen to which they are both attached,form a 3 to 7 membered heterocyclic ring wherein any one carbon atom ofsaid heterocyclic ring can optionally be replaced with —O—, —S—or—NR^(a)—. Therefore, by way of example and not limitation, the moietyNR¹¹R¹² can be represented by the heterocycles:

and the like.

In another embodiment, provided is a method of treating an Arenaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I-IV, whereineach R³, R⁴, R⁵, R⁶, R¹¹ or R¹² is, independently, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl or aryl(C₁-C₈)alkyl, wherein said(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or aryl(C₁-C₈)alkyl are,independently, optionally substituted with one or more halo, hydroxy,CN, N₃, N(R^(a))₂ or OR^(a). Therefore, by way of example and notlimitation, R³, R⁴, R⁵, R⁶, or R¹² could represent moieties such as—CH(NH₂)CH₃, —CH(OH)CH₂CH₃, —CH(NH₂)CH(CH₃)₂, —CH₂CF₃, —(CH₂)₂CH(N₃)CH₃,—(CH₂)₆NH₂ and the like.

In another embodiment, provided is a method of treating an Arenaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I-IV, whereinR³, R⁴, R⁵, R⁶, R¹¹ or R¹² is (C₁-C₈)alkyl wherein one or more of thenon-terminal carbon atoms of each said (C₁-C₈)alkyl may be optionallyreplaced with —O—, —S—or —NR^(a)—. Therefore, by way of example and notlimitation, R³, R⁴, R⁵, R⁶, R¹¹ or R¹² could represent moieties such as—CH₂OCH₃, —CH₂OCH₂CH₃, —CH₂OCH(CH₃)₂, —CH₂SCH₃, —(CH₂)₆OCH₃,—(CH₂)₆N(CH₃)₂ and the like.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula I, the compoundis

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula I, the compoundis

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, thecompound is:

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula IV, thecompound is:

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula I-IV, thecompound is

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating an Arenaviridaeinfection comprising administering a compound of Formula I-IV, thecompound is

or a pharmaceutically acceptable salt or ester thereof.

Provided is a method for treating a Coronaviridae infection in a humanin need thereof comprising administering a therapeutically effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein:    -   each R¹ is H or halogen;    -   each R², R³, R⁴ or R⁵ is independently H, OR^(a), N(R^(a))₂, N₃,        CN, NO₂, S(O)_(n)R^(a), halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl or        (C₂-C₈)substituted alkynyl;        -   or any two R², R³, R⁴ or R⁵ on adjacent carbon atoms when            taken together are —O(CO)O— or when taken together with the            ring carbon atoms to which they are attached form a double            bond;    -   R⁶ is OR^(a), N(R^(a))₂, N₃, CN, NO₂, S(O),R^(a), —C(═O)R¹¹,        —C(═O)OR¹¹, —C(═O)NR¹¹R¹²⁹, —C(═O)SR¹¹, —S(O)R¹¹, —S(O)₂R¹¹,        —S(O)(OR¹¹), —S(O)₂(OR¹¹), —SO₂NR¹¹R¹², halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl,        (C₂-C₈)substituted alkynyl, or (C₆-C₂₀)aryl(C₁-C₈)alkyl;    -   R⁷ is selected from a group consisting of        -   a) H, —C(═O)R¹¹, —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹,            —S(O)R¹¹, —S(O)₂R¹¹, —S(O)(OR¹¹), —S(O)₂(OR¹¹), or            —SO₂NR¹¹R¹²,            -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl,                (C₂-C₈)alkynyl or (C₆-C₂₀)aryl(C₁-C₈)alkyl of each R¹¹                or R¹² is, independently, optionally substituted with                one or more halo, hydroxy, CN, N₃, N(R^(a))₂ or OR^(a);                and wherein one or more of the non-terminal carbon atoms                of each said (C₁-C₈)alkyl may be optionally replaced                with —O—, —S— or —NR^(a)—,

-   -   -   -   wherein:                -   R^(c) is selected from phenyl, 1-naphthyl,                    2-naphthyl,

-   -   -   -   -   R^(d) is H or CH₃;                -   R^(e1) and R^(e2) are each independently H,                    (C₁-C₆)alkyl or benzyl;                -   R^(f) is selected from H, (C₁-C₈)alkyl, benzyl,                    (C₃-C₆)cycloalkyl, and —CH₂—(C₃-C₆)cycloalkyl;                -   R^(g) is selected from (C₁-C₈)alkyl,                    —O—(C₁-C₈)alkyl, benzyl, —O-benzyl,                    —CH₂—(C₃-C₆)cycloalkyl, —OCH₂—(C₃-C₆)cycloalkyl, and                    CF₃; and                -   n′ is selected from 1, 2, 3, and 4; and

        -   d) a group of the formula:

-   -   -   -   wherein:                -   Q is O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;                -   Z¹ and Z², when taken together, are                    —Q¹(C(R^(y))₂)₃Q¹-;                -   wherein                -    each Q¹ is independently O, S, or NR; and                -    each R^(y) is independently H, F, Cl, Br, I, OH, R,                    —C(═Q²)R, —C(═Q²)OR, —C(═Q²)N(R)₂, —N(R)₂, —⁺N(R)₃,                    —SR, —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR),                    —OC(═Q¹)R, —OC(═Q²)OR, —OC(═Q²)(N(R)₂), —SC(═Q²)R,                    —SC(═Q²)OR, —SC(═Q²)(N(R)₂), —N(R)C(═Q²)R,                    —N(R)C(═Q²)OR, —N(R)C(═Q²)N(R)₂, —SO₂NR₂, —CN, —N₃,                    —NO₂, —OR, or Z³; or when taken together, two R^(y)                    on the same carbon atom form a carbocyclic ring of 3                    to 7 carbon atoms;                -    each Q² is independently, O, S, NR, ⁺N(O)(R),                    N(OR), ⁺N(O)(OR), or N—NR₂; or                -   Z¹ and Z² are each, independently, a group of the                    Formula Ia:

-   -   -   -   -   wherein:                -    each Q³ is independently a bond, O, CR₂, NR,                    ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S, S—S, S(O), or                    S(O)₂;                -    M2 is 0, 1 or 2;                -    each R^(x) is independently R^(y) or the formula:

-   -   -   -   -    wherein:                -    each M1a, M1c, and M1d is independently 0 or 1;                -    M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;                -    Z³ is Z⁴ or Z⁵;                -    Z⁴ is R, —C(Q²)R^(y), —C(Q²)Z⁵, —SO₂R^(y), or                    —SO₂Z⁵; and                -    Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is                    independently substituted with 0 to 3 R^(y) groups;

    -   R⁸ is halogen, NR¹¹R¹², N(R¹¹)OR¹¹, NR¹¹NR¹¹R¹², N₃, NO, NO₂,        CHO, CN, —CH(═NR¹¹), —CH═NNHR¹¹, —CH═N(OR¹¹), —CH(OR¹¹)₂,        —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹², —C(═O)OR¹¹, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl,        (C₆-C₂₀)aryl(C₁-C₈)alkyl, OR¹¹ or SR¹¹;

    -   each R⁹ or R¹⁰ is independently H, halogen, NR¹¹R¹²,        NR¹¹NR¹¹R¹², N₃, NO, NO₂, CHO, CN, —CH(═NR¹¹), —CH═NHNR¹¹,        —CH═N(OR¹¹), —CH(OR¹¹)₂, —C(═O)NR¹¹R¹², —C(═S)NR¹¹R¹²,        —C(═O)OR¹¹, R¹¹, OR¹¹ or SR¹¹;

    -   each R¹¹ or R¹² is independently H, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        (C₆-C₂₀)optionally substituted aryl, optionally substituted        heteroaryl, —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl; or R¹¹ and R¹² taken together with a        nitrogen to which they are both attached form a 3 to 7 membered        heterocyclic ring wherein any one carbon atom of said        heterocyclic ring can optionally be replaced with —O—, —S—or        —NR^(a)—;

    -   each R^(a) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, —C(═O)R, —C(═O)OR, —C(═O)NR₂, —C(═O)SR,        —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), or —SO₂NR₂; wherein

    -   each R is independently H, (C₁-C₈) alkyl, (C₁-C₈) substituted        alkyl, (C₂-C₈)alkenyl, (C₂-C₈) substituted alkenyl, (C₂-C₈)        alkynyl, (C₂-C₈) substituted alkynyl, (C₆-C₂₀)aryl,        (C₆-C₂₀)substituted aryl, (C₂-C₂₀)heterocyclyl,        (C₂-C₂₀)substituted heterocyclyl, (C₆-C₂₀)aryl(C₁-C₈)alkyl or        substituted (C₆-C₂₀)aryl(C₁-C₈)alkyl;

    -   each n is independently 0, 1, or 2; and

    -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or        (C₆-C₂₀)aryl(C₁-C₈)alkyl of each R², R³, R⁵, R⁶, R¹¹ or R¹² is,        independently, optionally substituted with one or more halo,        hydroxy, CN, N₃, N(R^(a))₂ or OR^(a); and wherein one or more of        the non-terminal carbon atoms of each said (C₁-C₈)alkyl may be        optionally replaced with —O—, —S—or —NR^(a)—.

In another embodiment, provided is a method of treating a Coronaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I representedby Formula II:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein    -   R¹, R³, R⁵, R⁷, R⁸ and R⁹ are as defined above for Formula I;    -   each R² is OR^(a) or halogen; and    -   R⁶ is OR^(a), N(R^(a))₂, N₃, CN, S(O)_(n)R^(a), —C(═O)R¹¹,        —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹, —S(O)R¹¹, —S(O)₂R¹¹,        —S(O)(OR¹¹), —S(O)₂(OR¹¹), —SO₂NR¹¹R¹², halogen, (C₁-C₈)alkyl,        (C₄-C₈)carbocyclylalkyl, (C₁-C₈)substituted alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or        (C₂-C₈)substituted alkynyl.

In one embodiment of the method of treating a Coronaviridae infection byadministering a compound of Formula II, R¹ of Formula II is H. Inanother aspect of this embodiment R⁶ of Formula II is N₃, CN, halogen,(C₁-C₈)alkyl, (C₁-C₈)substituted alkyl, (C₂-C₈)alkenyl,(C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or (C₂-C₈)substitutedalkynyl. In another aspect of this embodiment, R⁶ of Formula II is CN,methyl, ethenyl, or ethynyl. In another aspect of this embodiment, R⁶ ofFormula II is CN. In another aspect of this embodiment, R⁶ of Formula IIis methyl. In another aspect of this embodiment, R⁵ of Formula II is H.In another aspect of this embodiment, R² of Formula II is OR^(a). Inanother aspect of this embodiment, R² of Formula II is OH. In anotheraspect of this embodiment, R² of Formula II is F. In another aspect ofthis embodiment, R³ of Formula II is OR^(a). In another aspect of thisembodiment, R³ of Formula II is OH, —OC(═O)R¹¹, or —OC(═O)OR¹¹. Inanother aspect of this embodiment, R³ of Formula II is OH. In anotheraspect of this embodiment, R⁸ of Formula II is NR¹¹R¹². In anotheraspect of this embodiment, R⁸ of Formula II is NH₂. In another aspect ofthis embodiment, R⁸ of Formula II is OR¹¹. In another aspect of thisembodiment, R⁸ of Formula II is OH. In another aspect of thisembodiment, R⁹ of Formula II is H. In another aspect of this embodiment,R⁹ of Formula II is NR¹¹R¹². In another aspect of this embodiment, R⁹ ofFormula II is NH₂. In another aspect of this embodiment, R⁷ of FormulaII is H, —C(═O)R¹¹, —C(═O)OR¹¹ or

In another aspect of this embodiment, R⁷ of Formula II is H. In anotheraspect of this embodiment, R⁷ of Formula II is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula II, theCoronaviridae infection is caused by a Coronaviridae virus. In anotheraspect of this embodiment, the Coronaviridae virus is a MERS virus orSARS virus. In another aspect of this embodiment, the Coronaviridaevirus is a MERS virus. In another aspect of this embodiment, theCoronaviridae virus is a SARS virus. In another aspect of thisembodiment, the Coronaviridae virus is caused by a MERS virus caused bya strain selected from known strains.

In another embodiment, provided is a method of treating a Coronaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I representedby Formula III:

or a pharmaceutically acceptable salt or ester, thereof;

-   -   wherein    -   R⁶, R⁷, R⁸ and R⁹ are as defined above for Formula II;    -   each R² is OR^(a) or F; and    -   each R³ is OR^(a).

In one embodiment of the method of treating a Coronaviridae infectioncomprising administering a compound of Formula III, R⁶ of Formula III isN₃, CN, halogen, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl, (C₂-C₈)alkenyl,(C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or (C₂-C₈)substitutedalkynyl. In another aspect of this embodiment, R⁶ of Formula III is CN,methyl, ethenyl, or ethynyl. In another aspect of this embodiment, R⁶ ofFormula III is CN. In another aspect of this embodiment, R⁶ of FormulaIII is methyl. In another aspect of this embodiment, R² of Formula IIIis OR^(a). In another aspect of this embodiment, R² of Formula III isOH. In another aspect of this embodiment, R² of Formula III is F. Inanother aspect of this embodiment, R³ of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R³ of Formula IIIis OH. In another aspect of this embodiment, R⁸ of Formula III isNR¹¹R¹². In another aspect of this embodiment, R⁸ of Formula III is NH₂.In another aspect of this embodiment, R⁸ of Formula III is OR¹¹. Inanother aspect of this embodiment, R⁸ of Formula III is OH. In anotheraspect of this embodiment, R⁹ of Formula III is H. In another aspect ofthis embodiment, R⁹ of Formula III is NR¹¹R¹². In another aspect of thisembodiment, R⁹ of Formula III is NH₂. In another aspect of thisembodiment, R⁷ of Formula III is H, —C(═O)R¹¹, —C(═O)OR¹¹ or

In another aspect of this embodiment, R⁷ of Formula III is H. In anotheraspect of this embodiment, R⁷ of Formula III is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula III, R⁶ ofFormula III is N₃, CN, halogen, (C₁-C₈)alkyl, (C₁-C₈)substituted alkyl,(C₂-C₈)alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈)alkynyl, or(C₂-C₈)substituted alkynyl and R⁸ is NH₂. In another aspect of thisembodiment, R⁶ of Formula III is CN, methyl, ethenyl, or ethynyl. Inanother aspect of this embodiment, R⁶ of Formula III is CN. In anotheraspect of this embodiment, R⁶ of Formula III is methyl. In anotheraspect of this embodiment, R² of Formula III is OR^(a). In anotheraspect of this embodiment, R² of Formula III is OH, —OC(═O)R¹¹, or—OC(═O)OR¹¹. In another aspect of this embodiment, R² of Formula III isOH. In another aspect of this embodiment, R² of Formula III is F. Inanother aspect of this embodiment, R³ of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R³ of Formula IIIis OH. In another aspect of this embodiment, R⁹ of Formula III is H. Inanother aspect of this embodiment, R⁹ of Formula III is NR¹¹R¹². Inanother aspect of this embodiment, R⁹ of Formula III is NH₂. In anotheraspect of this embodiment, R⁷ of Formula III is H, —C(═O)R¹¹, —C(═O)OR¹¹or

In another aspect of this embodiment, R⁷ of Formula III is H. In anotheraspect of this embodiment, R⁷ of Formula III is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula III, R⁶ ofFormula III is CN, methyl, ethenyl, or ethynyl, R⁸ is NH₂, and R⁹ is H.In another aspect of this embodiment, R⁶ of Formula III is CN. Inanother aspect of this embodiment, R⁶ of Formula III is methyl. Inanother aspect of this embodiment, R² of Formula III is OR^(a). Inanother aspect of this embodiment, R² of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R² of Formula IIIis OH. In another aspect of this embodiment, R² of Formula III is F. Inanother aspect of this embodiment, R³ of Formula III is OH, —OC(═O)R¹¹,or —OC(═O)OR¹¹. In another aspect of this embodiment, R³ of Formula IIIis OH. In another aspect of this embodiment, R⁷ of Formula III is H,—C(═O)R¹¹, —C(═O)OR¹¹ or

In another aspect of this embodiment, R⁷ of Formula III is H. In anotheraspect of this embodiment, R⁷ of Formula III is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula III, theCoronaviridae infection is caused by a Coronaviridae virus. In anotheraspect of this embodiment, the Coronaviridae virus is a MERS virus orSARS virus. In another aspect of this embodiment, the Coronaviridaevirus is a MERS virus. In another aspect of this embodiment, theCoronaviridae virus is a SARS virus. In another aspect of thisembodiment, the Coronaviridae virus is caused by a MERS virus caused bya strain selected from known strains.

In another embodiment, provided is a method of treating a Coronaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I representedby Formula IV:

or a pharmaceutically acceptable salt or ester, thereof;

-   wherein R⁷ is as defined above for Formula I.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ can beH. In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ isselected from the group of a), b), or c) as defined for Formula I.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

-   -   wherein Z¹ and Z² are each, independently, a group having the        structure:

and Z³ is Z⁵.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein Z¹ and Z² are each, independently, a group having the structure:

and Z³ is Z⁵.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein each Q^(3b) is, independently, O or N(R). In another embodiment,each Q^(3b) is O and each R^(x) is independently:

wherein M12c is 1, 2 or 3 and each Q³ is independently a bond, O, CR₂,or S.

In some embodiments, R^(e1) and R^(e2) can each independently be H,C₁-C₆ alkyl or benzyl. In some embodiments, R^(e1) can be H, C₁-C₆ alkylor benzyl, and R^(e2) can be H or C₁-C₆ alkyl. In some embodiments,R^(e1) and R^(e2) can each independently be H or C₁-C₆ alkyl. In someembodiments, R^(e1) and R^(e2) can each independently be H or benzyl. Insome embodiments, R^(e1) can be H, methyl or benzyl, and R^(e2) can be Hor methyl. In some embodiments, R^(e1) can be H or methyl, and R^(e2)can be H or methyl. In some embodiments, R^(e1) can be methyl, andR^(e2) can be H or methyl. In some embodiments, R^(e1) can be H orbenzyl, and R^(e2) can be H or methyl.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein R^(f) is selected from the group of from H, C₁-C₈ alkyl, benzyl,C₃-C₆ cycloalkyl, and —CH₂—C₃-C₆ cycloalkyl. In another embodiment of acompound of Formula IV, R^(f) is C₁-C₈ alkyl. In another embodiment of acompound of Formula IV, R^(f) is 2-ethylbutyl.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

-   -   wherein    -   R^(f) is selected from H, C₁-C₈ alkyl, benzyl, C₃-C₆ cycloalkyl,        and CH₂—C₃-C₆ cycloalkyl; and    -   R^(g) is selected from C₁-C₈ alkyl, —O—C₁-C₈ alkyl, benzyl,        —O-benzyl, —CH₂—C₃-C₆ cycloalkyl, —O—CH₂—C₃-C₆ cycloalkyl, and        CF₃.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

wherein R^(f) is selected from H, C₁-C₈ alkyl, benzyl, C₃-C₆ cycloalkyl,and —CH₂—C₃-C₆ cycloalkyl. In another embodiment of a compound ofFormula IV, R^(f)is C₁-C₈ alkyl. In another embodiment of a compound ofFormula IV, R^(f) is C₁-C₆ alkyl. In another embodiment of a compound ofFormula IV, R^(f) is 2-ethylbutyl.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is:

wherein R^(g) is selected from C₁-C₈ alkyl, —O—C₁-C₈ alkyl, benzyl,—O-benzyl, —CH₂—C₃-C₆ cycloalkyl, —O—CH₂—C₃-C₆ cycloalkyl, and CF₃. Inanother embodiment of a compound of Formula IV, R^(f) is C₁-C₈ alkyl. Inanother embodiment of a compound of Formula IV, R^(f) is C₁-C₆ alkyl.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ isselected from the group of:

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, R⁷ is

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, Z¹ and Z²can each be:

In another embodiment, provided is a method of treating a Coronaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formulas I-IV, whereinR¹¹ or R¹² is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl, optionally substituted aryl,optionally substituted heteroaryl, —C(═O)(C₁-C₈)alkyl,—S(O)_(n)(C₁-C₈)alkyl or aryl(C₁-C₈)alkyl. In another embodiment, R¹¹and R¹² taken together with a nitrogen to which they are both attached,form a 3 to 7 membered heterocyclic ring wherein any one carbon atom ofsaid heterocyclic ring can optionally be replaced with —O—, —S—or—NR^(a)—. Therefore, by way of example and not limitation, the moietyNR¹¹R¹² can be represented by the heterocycles:

and the like.

In another embodiment, provided is a method of treating a Coronaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I-IV, whereineach R³, R⁴, R⁵, R⁶, R¹¹ or R¹² is, independently, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl or aryl(C₁-C₈)alkyl, wherein said(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or aryl(C₁-C₈)alkyl are,independently, optionally substituted with one or more halo, hydroxy,CN, N₃, N(R^(a))₂ or OR^(a). Therefore, by way of example and notlimitation, R³, R⁴, R⁵, R⁶, R¹¹ or R¹² could represent moieties such as—CH(NH₂)CH₃, —CH(OH)CH₂CH₃, —CH(NH₂)CH(CH₃)₂, —CH₂CF₃, —(CH₂)₂CH(N₃)CH₃,—(CH₂)₆NH₂ and the like.

In another embodiment, provided is a method of treating a Coronaviridaeinfection in a human in need thereof comprising administering atherapeutically effective amount of a compound of Formula I-IV, whereinR³, R⁴, R⁵, R⁶, R¹¹ or R¹² is (C₁-C₈)alkyl wherein one or more of thenon-terminal carbon atoms of each said (C₁-C₈)alkyl may be optionallyreplaced with —O—, —S— or —NR^(a)—. Therefore, by way of example and notlimitation, R³, R⁴, R⁵, R⁶, R¹¹ or R¹² could represent moieties such as—CH₂OCH₃, —CH₂OCH₂CH₃, —CH₂OCH(CH₃)₂, —CH₂SCH₃, —(CH₂)₆OCH₃,—(CH₂)₆N(CH₃)₂ and the like.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula I, the compoundis

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula I, the compoundis

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, thecompound is:

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula IV, thecompound is:

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula I-IV, thecompound is

or a pharmaceutically acceptable salt or ester thereof.

In another embodiment of the method of treating a Coronaviridaeinfection comprising administering a compound of Formula I-IV, thecompound is

or a pharmaceutically acceptable salt or ester thereof.

Methods of treatment herein include those for treating coronavirusinfections in a human, including infections caused by alphacoronaviruses 229E (HCoV-229E) and NL63 (HCoV-NL63, New Havencoronavirus), beta coronaviruses OC43 (HCoV—OC43), HKU1, SARS-CoV (thecoronavirus responsible for Severe Acute Respiratory Syndrome, or SARS),and MERS-CoV (the coronavirus responsible for Middle East RespiratorySyndrome), previously known as Novel coronavirus 2012 and HCoV-EMC.

Names of compounds of the present disclosure are provided using ACD/Namesoftware for naming chemical compounds (Advanced Chemistry Development,Inc., Toronto, Canada). Other compounds or radicals may be named withcommon names or systematic or non-systematic names. The naming andnumbering of the compounds of the disclosure is illustrated with arepresentative compound of Formula I:

which is named (2S)-2-ethylbutyl2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate.Other compounds of the present invention include:

which is named (S)-2-ethylbutyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate,and

which is named (S)-2-ethylbutyl2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

Any reference to the compounds of the invention described herein alsoincludes a reference to a physiologically acceptable salt thereof.Examples of physiologically acceptable salts of the compounds of theinvention include salts derived from an appropriate base, such as analkali metal or an alkaline earth (for example, Na⁺, Li⁺, K⁺, Ca⁺² andMg⁺²), ammonium and NR₄ ⁺(wherein R is defined herein). Physiologicallyacceptable salts of a nitrogen atom or an amino group include (a) acidaddition salts formed with inorganic acids, for example, hydrochloricacid, hydrobromic acid, sulfuric acid, sulfamic acids, phosphoric acid,nitric acid and the like; (b) salts formed with organic acids such as,for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannicacid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid,malonic acid, sulfosalicylic acid, glycolic acid,2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalicacid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine,glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucineand the like; and (c) salts formed from elemental anions for example,chlorine, bromine, and iodine. Physiologically acceptable salts of acompound of a hydroxy group include the anion of said compound incombination with a suitable cation such as Na⁺ and NR₄ ⁺.

A compound of Formula I-IV and its pharmaceutically acceptable salts mayexist as different polymorphs or pseudopolymorphs. As used herein,crystalline polymorphism means the ability of a crystalline compound toexist in different crystal structures. The crystalline polymorphism mayresult from differences in crystal packing (packing polymorphism) ordifferences in packing between different conformers of the same molecule(conformational polymorphism). As used herein, crystallinepseudopolymorphism means the ability of a hydrate or solvate of acompound to exist in different crystal structures. The pseudopolymorphsof the instant invention may exist due to differences in crystal packing(packing pseudopolymorphism) or due to differences in packing betweendifferent conformers of the same molecule (conformationalpseudopolymorphism). The instant invention comprises all polymorphs andpseudopolymorphs of the compounds of Formula I-III and theirpharmaceutically acceptable salts.

A compound of Formula I-IV and its pharmaceutically acceptable salts mayalso exist as an amorphous solid. As used herein, an amorphous solid isa solid in which there is no long-range order of the positions of theatoms in the solid. This definition applies as well when the crystalsize is two nanometers or less. Additives, including solvents, may beused to create the amorphous forms of the instant invention. The instantinvention comprises all amorphous forms of the compounds of Formula I-IVand their pharmaceutically acceptable salts.

For therapeutic use, salts of active ingredients of the compounds of theinvention will be physiologically acceptable, i.e. they will be saltsderived from a physiologically acceptable acid or base. However, saltsof acids or bases which are not physiologically acceptable may also finduse, for example, in the preparation or purification of aphysiologically acceptable compound. All salts, whether or not derivedform a physiologically acceptable acid or base, are within the scope ofthe present invention.

Finally, it is to be understood that the compositions herein comprisecompounds of the invention in their un-ionized, as well as zwitterionicform, and combinations with stoichiometric amounts of water as inhydrates.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs of compounds withinthe scope of Formula I-IV and pharmaceutically acceptable salts thereofare embraced by the present invention. All mixtures of such enantiomersand diastereomers are within the scope of the present invention.

The compounds of the invention, exemplified by Formula I-IV may havechiral centers, e.g. chiral carbon or phosphorus atoms. The compounds ofthe invention thus include racemic mixtures of all stereoisomers,including enantiomers, diastereomers, and atropisomers. In addition, thecompounds of the invention include enriched or resolved optical isomersat any or all asymmetric, chiral atoms. In other words, the chiralcenters apparent from the depictions are provided as the chiral isomersor racemic mixtures. Both racemic and diastereomeric mixtures, as wellas the individual optical isomers isolated or synthesized, substantiallyfree of their enantiomeric or diastereomeric partners, are all withinthe scope of the invention. The racemic mixtures are separated intotheir individual, substantially optically pure isomers throughwell-known techniques such as, for example, the separation ofdiastereomeric salts formed with optically active adjuncts, e.g., acidsor bases followed by conversion back to the optically active substances.In most instances, the desired optical isomer is synthesized by means ofstereospecific reactions, beginning with the appropriate stereoisomer ofthe desired starting material.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and 1, D and L, or (+) and (−) areemployed to designate the sign of rotation of plane-polarized light bythe compound, with S, (−), or 1 meaning that the compound islevorotatory while a compound prefixed with R, (+), or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The compounds of the invention can also exist as tautomeric isomers incertain cases. Although only one delocalized resonance structure may bedepicted, all such forms are contemplated within the scope of theinvention. For example, ene-amine tautomers can exist for purine,pyrimidine, imidazole, guanidine, amidine, and tetrazole systems and alltheir possible tautomeric forms are within the scope of the invention.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent unlabeled forms as well as isotopicallylabeled forms of the compounds. Isotopically labeled compounds havestructures depicted by the formulas given herein except that one or moreatoms are replaced by an atom having a selected atomic mass or massnumber. Examples of isotopes that can be incorporated into compounds ofthe disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵Cl,³⁶Cl and ¹²⁵I. Various isotopically labeled compounds of the presentdisclosure, for example those into which radioactive isotopes such as³H, ¹³C and ¹⁴C are incorporated. Such isotopically labelled compoundsmay be useful in metabolic studies, reaction kinetic studies, detectionor imaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays or in radioactive treatment ofpatients.

The disclosure also included compounds of Formula I in which from 1 to nhydrogens attached to a carbon atom is/are replaced by deuterium, inwhich n is the number of hydrogens in the molecule. Such compoundsexhibit increased resistance to metabolism and are thus useful forincreasing the half-life of any compound of Formula I when administeredto a mammal, particularly a human. See, for example, Foster, “DeuteriumIsotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci.5(12):524-527 (1984). Such compounds are synthesized by means well knownin the art, for example by employing starting materials in which one ormore hydrogens have been replaced by deuterium.

Deuterium labeled or substituted therapeutic compounds of the disclosuremay have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life, reduced dosage requirements and/oran improvement in therapeutic index. An ¹⁸F labeled compound may beuseful for PET or SPECT studies. Isotopically labeled compounds of thisdisclosure and prodrugs thereof can generally be prepared by carryingout the procedures disclosed in the schemes or in the examples andpreparations described below by substituting a readily availableisotopically labeled reagent for a non-isotopically labeled reagent. Itis understood that deuterium in this context is regarded as asubstituent in the compound of Formula I.

The concentration of such a heavier isotope, specifically deuterium, maybe defined by an isotopic enrichment factor. In the compounds of thisdisclosure any atom not specifically designated as a particular isotopeis meant to represent any stable isotope of that atom. Unless otherwisestated, when a position is designated specifically as “H” or “hydrogen”,the position is understood to have hydrogen at its natural abundanceisotopic composition. Accordingly, in the compounds of this disclosureany atom specifically designated as a deuterium (D) is meant torepresent deuterium.

Whenever a compound described herein is substituted with more than oneof the same designated group, e.g., “R” or “R”, then it will beunderstood that the groups may be the same or different, i.e., eachgroup is independently selected. Wavy lines,

, indicate the site of covalent bond attachments to the adjoiningsubstructures, groups, moieties, or atoms. Selected substituentscomprising the compounds of Formula I-IV are present to a recursivedegree. In this context, “recursive substituent” means that asubstituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number ofcompounds may be present in any given embodiment. For example, R^(x)comprises a R^(y) substituent. R^(y) can be R. R can be Z³. Z³ can be Z⁴and Z⁴ can be R or comprise substituents comprising R^(y).Alternatively, Z³ can be Z⁵ which can comprise substituents comprisingB^(y). One of ordinary skill in the art of medicinal chemistryunderstands that the total number of such substituents is reasonablylimited by the desired properties of the compound intended. Suchproperties include, by way of example and not limitation, physicalproperties such as molecular weight, solubility or log P, applicationproperties such as activity against the intended target, and practicalproperties such as ease of synthesis.

By way of example and not limitation, Z³ and R^(y) are recursivesubstituents in certain embodiments. Typically, each recursivesubstituent can independently occur 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0, times in a given embodiment.More typically, each recursive substituent can independently occur 12 orfewer times in a given embodiment. Even more typically, each recursivesubstituent can independently occur 3 or fewer times in a givenembodiment. For example, Z³ will occur 0 to 8 times, R^(y) will occur 0to 6 times in a given embodiment. Even more typically, Z³ will occur 0to 6 times and R^(y) will occur 0 to 4 times in a given embodiment.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal chemistry understands theversatility of such substituents. To the degree that recursivesubstituents are present in an embodiment of the invention, the totalnumber will be determined as set forth above.

The compounds of the present invention can be prepared by methods knownto one of skill in the art. For example, the compounds of the presentinvention can be prepared according to the methods described in U.S.Pat. No. 8,008,264 and U.S. Application Publication No. US 2012/0027752.

A. Substituted Forms of the Compounds

The compounds of the Formula I-IV may comprise a phosphate group as R⁷,R⁷ is selected from the group of

-   -   a) H, —C(═O)R¹¹, —C(═O)OR¹¹, —C(═O)NR¹¹R¹², —C(═O)SR¹¹,        —S(O)R¹¹, —S(O)₂R¹¹, —S(O)(OR¹¹), —S(O)₂(OR¹¹), SO₂NR¹¹R¹²    -   wherein    -   each R¹¹ or R¹² is independently H, (C₁-C₈)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₄-C₈)carbocyclylalkyl,        optionally substituted aryl, optionally substituted heteroaryl,        —C(═O)(C₁-C₈)alkyl, —S(O)_(n)(C₁-C₈)alkyl or aryl(C₁-C₈)alkyl;        or R¹¹ and R¹² taken together with a nitrogen to which they are        both attached form a 3 to 7 membered heterocyclic ring wherein        any one carbon atom of said heterocyclic ring can optionally be        replaced with —O—, —S—or —NR^(a)—;    -   each R^(a) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, aryl(C₁-C₈)alkyl, (C₄-C₈)carbocyclylalkyl,        —C(═O)R, —C(═O)OR, —C(═O)NR₂, —C(═O)SR, —S(O)R, —S(O)₂R,        —S(O)(OR), —S(O)₂(OR), or —SO₂NR₂;    -   wherein each R is independently H, (C₁-C₈) alkyl, (C₁-C₈)        substituted alkyl, (C₂-C₈)alkenyl, (C₂-C₈) substituted alkenyl,        (C₂-C₈) alkynyl, (C₂-C₈) substituted alkynyl, C₆-C₂₀ aryl,        C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocyclyl, C₂-C₂₀ substituted        heterocyclyl, arylalkyl or substituted arylalkyl; and    -   wherein each (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or        aryl(C₁-C₈)alkyl of each R¹¹ or R¹² is, independently,        optionally substituted with one or more halo, hydroxy, CN, N₃,        N(R^(a))₂ or OR^(a); and wherein one or more of the non-terminal        carbon atoms of each said (C₁-C₈)alkyl may be optionally        replaced with —O—, —S—or —NR^(a)—,

-   -   wherein:        -   R^(c) is selected from phenyl, 1-naphthyl, 2-naphthyl,

-   -   -   R^(d) is H or CH₃;        -   R^(e1) and R^(e2) are each independently H, C₁-C₆ alkyl or            benzyl;        -   R^(f) is selected from H, C₁-C₈ alkyl, benzyl, C₃-C₆            cycloalkyl, and CH₂—C₃-C₆ cycloalkyl;        -   R^(g) is selected from C₁-C₈ alkyl, —O—C₁-C₈ alkyl, benzyl,            —O-benzyl, —CH₂—C₃-C₆ cycloalkyl, —O—CH₂—C₃-C₆ cycloalkyl,            and CF₃; and        -   n′ is selected from 1, 2, 3, and 4; and

    -   d) a group of the formula:

-   -   wherein    -   Q is O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;    -   Z¹ and Z², when taken together, are —Q¹(C(R^(y))₂)₃Q¹-;    -   wherein        -   each Q¹ is independently O, S, or NR; and        -   each R^(y) is independently H, F, Cl, Br, I, OH, R,            —C(═Q²)R, —C(═Q²)OR, —C(═Q²)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR,            —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), —OC(═Q²)R,            —OC(═Q²)OR, —OC(═Q²)(N(R)₂), —SC(═Q²)R, —SC(═Q²)OR,            —SC(═Q²)(N(R)₂), —N(R)C(═Q²)R, —N(R)C(═Q²)OR,            —N(R)C(═Q²)N(R)₂, —SO₂NR₂, —CN, —N₃, —NO₂, —OR, or Z³; or            when taken together, two R^(y) on the same carbon atom form            a carbocyclic ring of 3 to 7 carbon atoms;        -   each Q² is independently, O, S, NR, ⁺N(O)(R), N(OR),            ⁺N(O)(OR), or N—NR₂; or    -   Z¹ and Z² are each, independently, a group of the Formula Ia:

-   -   wherein:        -   each Q³ is independently a bond, O, CR₂, NR, ⁺N(O)(R),            N(OR), ⁺N(O)(OR), N—NR₂, S, S—S, S(O), or S(O)₂;        -   M2 is 0, 1 or 2;        -   each R^(x) is independently R^(y) or the formula:

-   -   -   wherein:            -   each M1a, M1c, and M1d is independently 0 or 1;            -   M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;            -   Z³ is Z⁴ or Z⁵;            -   Z⁴ is R, —C(Q²)R^(y), —C(Q²)Z⁵, —SO₂R^(y), or —SO₂Z⁵;                and            -   Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is                independently substituted with 0 to 3 R^(y) groups.

Z⁵ carbocycles and Z⁵ heterocycles may be independently substituted with0 to 3 R^(y) groups. Z⁵ may be a saturated, unsaturated or aromatic ringcomprising a mono- or bicyclic carbocycle or heterocycle. Z⁵ may have 3to 10 ring atoms, e.g., 3 to 7 ring atoms. The Z⁵ rings are saturatedwhen containing 3 ring atoms, saturated or mono-unsaturated whencontaining 4 ring atoms, saturated, or mono- or di-unsaturated whencontaining 5 ring atoms, and saturated, mono- or di-unsaturated, oraromatic when containing 6 ring atoms.

A Z⁵ heterocycle may be a monocycle having 3 to 7 ring members (2 to 6carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or abicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S). Z⁵ heterocyclic monocyclesmay have 3 to 6 ring atoms (2 to 5 carbon atoms and 1 to 2 heteroatomsselected from N, O, and S); or 5 or 6 ring atoms (3 to 5 carbon atomsand 1 to 2 heteroatoms selected from N and S). Z⁵ heterocyclic bicycleshave 7 to 10 ring atoms (6 to 9 carbon atoms and 1 to 2 heteroatomsselected from N, O, and S) arranged as a bicyclo [4,5], [5,5], [5,6], or[6,6] system; or 9 to 10 ring atoms (8 to 9 carbon atoms and 1 to 2hetero atoms selected from N and S) arranged as a bicyclo [5,6] or [6,6]system. The Z⁵ heterocycle may be bonded to Q² through a carbon,nitrogen, sulfur or other atom by a stable covalent bond.

Z⁵ heterocycles include for example, pyridyl, dihydropyridyl isomers,piperidine, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl,imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, furanyl,thiofuranyl, thienyl, and pyrrolyl. Z⁵ also includes, but is not limitedto, examples such as:

Z⁵ carbocycles and heterocycles may be independently substituted with 0to 3 R groups, as defined above. For example, substituted Z⁵ carbocyclesinclude:

Examples of substituted phenyl carbocycles include:

In another embodiment, Z⁵ of the compounds of Formula I-IV is acarbocycle or a heterocycle wherein Z⁵ is independently substituted with0 to 3 R^(z) groups, wherein each R^(z) is independently H, F, Cl, Br,I, OH, R, —C(═Q²)R, —C(═Q²)OR, —C(═Q²)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR,—S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), —OC(═Q¹)R, —OC(═Q²)OR,—OC(═Q²)(N(R)₂), —SC(═Q²)R, —SC(═Q²)OR, —SC(═Q²)(N(R)₂), —N(R)C(═Q²)R,—N(R)C(═Q²)OR, —N(R)C(═Q²)N(R)₂, —SO₂NR₂, —CN, —N₃, —NO₂, or —OR.

Embodiments of

of Formula I-IV compounds include substructures such as:

wherein each Q^(3b) is, independently, O or N(R). In another aspect ofthis embodiment, each Q^(3b) is O and each R^(x) is independently:

wherein M12c is 1, 2 or 3 and each Q³ is independently a bond, O, CR₂,or S. In another aspect of this embodiment, one Q^(3b)—R^(x) is NH(R)and the other Q^(3b)—R^(x) is O—R^(x) wherein R^(x) is:

wherein M12c is 2. In another aspect of this embodiment, each Q^(3b) isO and each R^(x) is independently:

wherein M12c is 2. In another aspect of this embodiment, each Q^(3b) isO and each R^(x) is independently:

wherein M12c is 1 and Q³ is a bond, O, or CR₂.

Other embodiments of

of Formulas I-IV compounds include substructures such as:

wherein each Q³ is, independently, O or N(R). In another aspect of thisembodiment, each Q³ is O. In another aspect of this embodiment, thesubstructure is:

wherein R^(y) is Z⁵ as defined herein.

Another embodiment of

of Formula I-IV includes the substructures:

wherein each Q^(2c) is, independently, O, N(R^(y)) or S.

Another embodiment of

of Formula I-IV compounds includes the substructures wherein one of Z¹or Z² together with either R³ or R⁴ is —Q³— and the other of Z¹ or Z² isFormula Ia. Such an embodiment is represented by a compound of FormulaIb selected from:

In another aspect of the embodiment of Formula Ib, each Q and Q³ is O.In another aspect of the embodiment of Formula Ib, Z¹ or Z² isQ^(3b)—R^(x); each Q, Q³ and Q^(3b) is O and R^(x) is:

wherein M12c is 1, 2 or 3 and each Q³ is independently a bond, O, CR₂,or S. In another aspect of the embodiment of Formula Ib, Z¹ or Z² isQ^(3b)—R^(x); each Q, Q³ and Q^(3b) is O and R^(x) is:

wherein M12c is 2. In another aspect of the embodiment of Formula Ib, Z¹or Z² is Q^(3b)—R^(x); each Q, Q³ and Q^(3b) is O and R^(x) is:

wherein M12c is 1 and Q³ is a bond, O, or CR₂.

Another embodiment of

of Formula I-IV compounds includes a sub structure:

wherein Z⁵ is a carbocycle such as phenyl or substituted phenyl. Inanother aspect of this embodiment, the substructure is:

wherein Q^(3b) is O or N(R) and the phenyl carbocycle is substitutedwith 0 to 3 R groups. In another aspect of this embodiment of thesubstructure, R^(x) is:

wherein M12c is 1, 2 or 3 and each Q³ is independently a bond, O, CR₂,or S.

Another embodiment of

of Formula I-IV includes substructures:

The chiral carbon of the amino acid and lactate moieties may be eitherthe R or S configuration or the racemic mixture.

Another embodiment of

of Formula I-IV is substructure

wherein each Q³ is, independently, —O— or —NH—. In another aspect ofthis embodiment, R^(y) is (C₁-C₈) alkyl, (C₁-C₈) substituted alkyl,(C₂-C₈) alkenyl, (C₂-C₈) substituted alkenyl, (C₂-C₈) alkynyl or (C₂-C₈)substituted alkynyl. In another aspect of this embodiment, R^(y) is(C₁-C₈) alkyl, (C₁-C₈) substituted alkyl, (C₂-C₈) alkenyl, (C₂-C₈)substituted alkenyl, (C₂-C₈) alkynyl or (C₂-C₈) substituted alkynyl; andR is CH₃. In another aspect of this embodiment, R^(y) is (C₁-C₈) alkyl,(C₁-C₈) substituted alkyl, (C₂-C₈) alkenyl, (C₂-C₈) substituted alkenyl,(C₂-C₈) alkynyl or (C₂-C₈) substituted alkynyl; R is CH₃; and each Q³ is—NH—. In another aspect of this embodiment, Z¹ and Z² are,independently, nitrogen-linked, naturally occurring amino acids ornaturally occurring amino acid esters. In another aspect of thisembodiment, Z¹ and Z² are, independently, naturally-occurring 2-hydroxycarboxylic acids or naturally-occurring 2-hydroxy carboxylic acid esterswherein the acid or ester is linked to P through the 2-hydroxy group.

Another embodiment of

of Formula I-IV is substructure:

In one aspect of this embodiment, each R^(x) is, independently, (C₁-C₈)alkyl. In another aspect of this embodiment, each R^(x) is,independently, C₆-C₂₀ aryl or C₆-C₂₀ substituted aryl.

In a preferred embodiment,

is selected from

Embodiments of R^(x) include esters, carbamates, carbonates, thioesters,amides, thioamides, and urea groups:

B. Metabolites of the Compounds of the Invention

Also falling within the scope of this invention are the in vivometabolic products of the compounds described herein, to the extent suchproducts are novel and unobvious over the prior art. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof. Such products typically areidentified by preparing a radiolabelled (e.g. ¹⁴C or ³H) compound of theinvention, administering it parenterally in a detectable dose (e.g.greater than about 0.5 mg/kg) to an animal such as rat, mouse, guineapig, monkey, or to man, allowing sufficient time for metabolism to occur(typically about 30 seconds to 30 hours) and isolating its conversionproducts from the urine, blood or other biological samples. Theseproducts are easily isolated since they are labeled (others are isolatedby the use of antibodies capable of binding epitopes surviving in themetabolite). The metabolite structures are determined in conventionalfashion, e.g. by MS or NMR analysis. In general, analysis of metabolitesis done in the same way as conventional drug metabolism studieswell-known to those skilled in the art. The conversion products, so longas they are not otherwise found in vivo, are useful in diagnostic assaysfor therapeutic dosing of the compounds of the invention even if theypossess no anti arenaviridae activity of their own.

Recipes and methods for determining stability of compounds in surrogategastrointestinal secretions are known. Compounds are defined herein asstable in the gastrointestinal tract where less than about 50 molepercent of the protected groups are deprotected in surrogate intestinalor gastric juice upon incubation for 1 hour at 37° C. Simply because thecompounds are stable to the gastrointestinal tract does not mean thatthey cannot be hydrolyzed in vivo. The prodrugs of the inventiontypically will be stable in the digestive system but may besubstantially hydrolyzed to the parental drug in the digestive lumen,liver or other metabolic organ, or within cells in general.

III. PHARMACEUTICAL FORMULATIONS

The compounds of this invention are formulated with conventionalcarriers and excipients, which will be selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, bindersand the like. Aqueous formulations are prepared in sterile form, andwhen intended for delivery by other than oral administration generallywill be isotonic. All formulations will optionally contain excipientssuch as those set forth in the “Handbook of Pharmaceutical Excipients”(1986). Excipients include ascorbic acid and other antioxidants,chelating agents such as EDTA, carbohydrates such as dextran,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and thelike. The pH of the formulations ranges from about 3 to about 11, but isordinarily about 7 to 10. In some embodiments, the pH of theformulations ranges from about 2 to about 5, but is ordinarily about 3to 4.

While it is possible for the active ingredients to be administered aloneit may be preferable to present them as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the inventioncomprise at least one active ingredient, as above defined, together withone or more acceptable carriers therefor and optionally othertherapeutic ingredients, particularly those additional therapeuticingredients as discussed herein. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and physiologically innocuous to the recipient thereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

For infections of the eye or other external tissues e.g. mouth and skin,the formulations are preferably applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethyl sulphoxide andrelated analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate. Further emulgents and emulsion stabilizers suitable foruse in the formulation of the invention include Tween® 80.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. The cream should preferablybe a non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils are used.

Pharmaceutical formulations according to the present invention comprisea combination according to the invention together with one or morepharmaceutically acceptable carriers or excipients and optionally othertherapeutic agents. Pharmaceutical formulations containing the activeingredient may be in any form suitable for the intended method ofadministration. When used for oral use for example, tablets, troches,lozenges, aqueous or oil suspensions, dispersible powders or granules,emulsions, hard or soft capsules, syrups or elixirs may be prepared.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation. Tabletscontaining the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniquesincluding microencapsulation to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally-occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.Further non-limiting examples of suspending agents include Cyclodextrinand Captisol (=Sulfobutyl ether beta-cyclodextrin; SEB-beta-CD).

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally-occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution isotonic sodium chloride solution,and hypertonic sodium chloride solution.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to10%, and particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns, such as0.5, 1, 30, 35 etc., which is administered by rapid inhalation throughthe nasal passage or by inhalation through the mouth so as to reach thealveolar sacs. Suitable formulations include aqueous or oily solutionsof the active ingredient. Formulations suitable for aerosol or drypowder administration may be prepared according to conventional methodsand may be delivered with other therapeutic agents such as compoundsheretofore used in the treatment or prophylaxis of Arenaviridaeinfections as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Compounds of the invention are used to provide controlled releasepharmaceutical formulations containing as active ingredient one or morecompounds of the invention (“controlled release formulations”) in whichthe release of the active ingredient are controlled and regulated toallow less frequency dosing or to improve the pharmacokinetic ortoxicity profile of a given active ingredient.

IV. ROUTES OF ADMINISTRATION

One or more compounds of the invention (herein referred to as the activeingredients) are administered by any route appropriate to the conditionto be treated. Suitable routes include oral, rectal, nasal, pulmonary,topical (including buccal and sublingual), vaginal and parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intrathecal and epidural), and the like. It will be appreciated that thepreferred route may vary with for example the condition of therecipient. An advantage of the compounds of this invention is that theyare orally bioavailable and can be dosed orally.

In the methods of the present invention for the treatment ofArenaviridae infection, the compounds of the present invention can beadministered at any time to a human who may come into contact withhumans suffering from Arenaviridae infection or is already sufferingfrom Arenaviridae infection. In some embodiments, the compounds of thepresent invention can be administered prophylactically to humans cominginto contact with humans suffering from Arenaviridae infection. In someembodiments, administration of the compounds of the present inventioncan be to humans testing positive for Arenaviridae infection but not yetshowing symptoms of Arenaviridae infection. In some embodiments,administration of the compounds of the present invention can be tohumans upon commencement of symptoms of Arenaviridae infection.

Effective dose of active ingredient depends at least on the nature ofthe condition being treated, toxicity, whether the compound is beingused prophylactically (lower doses) or against an active viralinfection, the method of delivery, and the pharmaceutical formulation,and will be determined by the clinician using conventional doseescalation studies. It can be expected to be from about 0.0001 to about100 mg/kg body weight per day; typically, from about 0.01 to about 10mg/kg body weight per day; more typically, from about 0.01 to about 5mg/kg body weight per day; most typically, from about 0.05 to about 0.5mg/kg body weight per day. For example, the daily candidate dose for anadult human of approximately 70 kg body weight will range from 1 mg to1000 mg, preferably between 5 mg and 500 mg, and may take the form ofsingle or multiple doses.

The effective dose of a compound of the present invention for treatingthe Arenaviridae infection can depend on whether the dose is to be usedprophylactically or to treat a human already suffering from Arenaviridaeinfection. Moreover, the dose can depend on whether the human sufferingfrom Arenaviridae infection does not yet show symptoms or is alreadyshowing symptoms of Arenaviridae infection. Larger doses may benecessary for treating humans testing positive for Arenaviridaeinfection and for humans showing symptoms of Arenaviridae infection ascompared to humans receiving prophylactic treatment.

Any suitable period of time for administration of the compounds of thepresent invention is contemplated. For example, administration can befor from 1 day to 100 days, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, or 90 days. The administration can alsobe for from 1 week to 15 weeks, including 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 weeks. Longer periods of administration are alsocontemplated. The time for administration can depend on whether thecompound is being administered prophylactically or to treat a humansuffering from an Arenaviridae infection. For example, a prophylacticadministration can be for a period of time while the human is in regularcontact with other humans suffering from an Arenaviridae infection, andfor a suitable period of time following the last contact with a humansuffering from an Arenaviridae infection. For humans already sufferingfrom an Arenaviridae infection, the period of administration can be forany length of time necessary to treat the patient and a suitable periodof time following a negative test for Arenaviridae infection to ensurethe Arenaviridae infection does not return.

V. COMBINATION THERAPY

Compositions of the invention are also used in combination with otheractive ingredients. For the treatment of Arenaviridae virus infections,preferably, the other active therapeutic agent is active againstArenaviridae virus infections, particularly Lassa virus and Junin virusinfections. Non-limiting examples of these other active therapeuticagents are ribavirin, favipiravir (also known as T-705 or Avigan),T-705monophosphate, T-705 diphosphate, T-705 triphosphate, ST-193, andmixtures thereof. The compounds and compositions of the presentinvention are also intended for use with general care provided patientswith Arenaviridae viral infections, including parenteral fluids(including dextrose saline and Ringer's lactate) and nutrition,antibiotic (including metronidazole and cephalosporin antibiotics, suchas ceftriaxone and cefuroxime) and/or antifungal prophylaxis, fever andpain medication, antiemetic (such as metoclopramide) and/orantidiarrheal agents, vitamin and mineral supplements (including VitaminK and zinc sulfate), anti-inflammatory agents (such as ibuprofen), painmedications, and medications for other common diseases in the patientpopulation, such anti-malarial agents (including artemether andartesunate-lumefantrine combination therapy), typhoid (includingquinolone antibiotics, such as ciprofloxacin, macrolide antibiotics,such as azithromycin, cephalosporin antibiotics, such as ceftriaxone, oraminopenicillins, such as ampicillin), or shigellosis.

It is also possible to combine any compound of the invention with one ormore additional active therapeutic agents in a unitary dosage form forsimultaneous or sequential administration to a patient. The combinationtherapy may be administered as a simultaneous or sequential regimen.When administered sequentially, the combination may be administered intwo or more administrations.

Co-administration of a compound of the invention with one or more otheractive therapeutic agents generally refers to simultaneous or sequentialadministration of a compound of the invention and one or more otheractive therapeutic agents, such that therapeutically effective amountsof the compound of the invention and one or more other activetherapeutic agents are both present in the body of the patient.

Co-administration includes administration of unit dosages of thecompounds of the invention before or after administration of unitdosages of one or more other active therapeutic agents, for example,administration of the compounds of the invention within seconds,minutes, or hours of the administration of one or more other activetherapeutic agents. For example, a unit dose of a compound of theinvention can be administered first, followed within seconds or minutesby administration of a unit dose of one or more other active therapeuticagents. Alternatively, a unit dose of one or more other therapeuticagents can be administered first, followed by administration of a unitdose of a compound of the invention within seconds or minutes. In somecases, it may be desirable to administer a unit dose of a compound ofthe invention first, followed, after a period of hours (e.g., 1-12hours), by administration of a unit dose of one or more other activetherapeutic agents. In other cases, it may be desirable to administer aunit dose of one or more other active therapeutic agents first,followed, after a period of hours (e.g., 1-12 hours), by administrationof a unit dose of a compound of the invention.

The combination therapy may provide “synergy” and “synergistic”, i.e.the effect achieved when the active ingredients used together is greaterthan the sum of the effects that results from using the compoundsseparately. A synergistic effect may be attained when the activeingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by some other regimen.When delivered in alternation therapy, a synergistic effect may beattained when the compounds are administered or delivered sequentially,e.g. in separate tablets, pills or capsules, or by different injectionsin separate syringes. In general, during alternation therapy, aneffective dosage of each active ingredient is administered sequentially,i.e. serially, whereas in combination therapy, effective dosages of twoor more active ingredients are administered together. A synergisticanti-viral effect denotes an antiviral effect which is greater than thepredicted purely additive effects of the individual compounds of thecombination.

In still yet another embodiment, the present application provides formethods of inhibiting Arenaviridae polymerase in a cell, comprising:contacting a cell infected with an arenavirus with an effective amountof a compound of Formula I-IV, or a pharmaceutically acceptable salt,solvate, and/or ester thereof, whereby Arenaviridae polymerase isinhibited.

In still yet another embodiment, the present application provides formethods of inhibiting Arenaviridae polymerase in a cell, comprising:contacting a cell infected with arenavirus with an effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,solvate, and/or ester thereof, and at least one additional activetherapeutic agent, whereby Arenaviridae polymerase is inhibited.

In still yet another embodiment, the present application provides formethods of inhibiting Arenaviridae polymerase in a cell, comprising:contacting a cell infected with Arenaviridae virus with an effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, solvate, and/or ester thereof, and at least one additional activetherapeutic agent selected

In still yet another embodiment, the present application provides formethods of treating Arenaviridae virus infection in a human, comprising:administering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,solvate, and/or ester thereof.

In still yet another embodiment, the present application provides formethods of treating Arenaviridae virus infection in a human, comprising:administering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,solvate, and/or ester thereof, and at least one additional activetherapeutic agent, whereby Arenaviridae polymerase is inhibited.

In still yet another embodiment, the present application provides formethods of treating Arenaviridae virus infection in a human, comprising:administering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,solvate, and/or ester thereof, and at least one additional activetherapeutic agent.

Also provided is a kit that includes a compound of Formula I, or apharmaceutically acceptable salt, pharmaceutically acceptable ester,stereoisomer, mixture of stereoisomers or tautomer thereof. In separateembodiments individual kits are provided includes a compound selectedfrom the group of each of the Formulas herein, as well as each subgroupand embodiment thereof, including Formula II, Formula II, Formula IV,and individual Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and32 (Compounds 1-32), or a pharmaceutically acceptable salt,pharmaceutically acceptable ester, stereoisomer, mixture ofstereoisomers or tautomer thereof. In one aspect, the kit comprises acompound of Formula I, or a pharmaceutically acceptable salt thereof.Each of the individual kits described herein may comprise a label and/orinstructions for use of the compound in the treatment of a disease orcondition in a subject (e.g., human) in need thereof. In someembodiments, the disease or condition is a human Arenaviridae viralinfection, including a Lassa viral infection or a Junin viral infection.In other embodiments, each separate kit may also contain instructionsfor use of additional medical agents in combination with the compound ofFormula I in the treatment of a disease or condition in a subject (e.g.,human) in need thereof. In certain of these embodiments, the disease orcondition is a human Arenaviridae viral infection, including a Lassaviral infection or a Junin viral infection. In each of the kits hereinthere is a further embodiment in which the kit comprises individual doseunits of a compound as described herein, or a pharmaceuticallyacceptable salt, racemate, enantiomer, diastereomer, tautomer,polymorph, pseudopolymorph, amorphous form, hydrate or solvate thereof.Examples of individual dosage units may include pills, tablets,capsules, prefilled syringes or syringe cartridges, IV bags, etc., eachcomprising a therapeutically effective amount of the compound inquestion, or a pharmaceutically acceptable salt, racemate, enantiomer,diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form,hydrate or solvate thereof. In some embodiments, the kit may contain asingle dosage unit and in others multiple dosage units are present, suchas the number of dosage units required for a specified regimen orperiod.

Also provided are articles of manufacture that include a compound ofFormula I, or a pharmaceutically acceptable salt, pharmaceuticallyacceptable ester, stereoisomer, mixture of stereoisomers or tautomerthereof and a container. In one aspect, the article of manufacturecomprises a compound of Formula I, Formula II, Formula II, Formula IV,and individual Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and32 (Compounds 1-32), or a pharmaceutically acceptable salt thereof, anda container. In separate embodiments, the container of the article ofmanufacture may be a vial, jar, ampoule, preloaded syringe, blisterpackage, tin, can, bottle, box, or an intravenous bag.

Also provided as separate embodiments are the uses of a compoundselected from each of the Formulas herein, as well as each subgroup andembodiment thereof, including a compound selected from the group ofFormula (I), Formula (II), Formula (III), Formula (IV), or one of thespecific compounds of the examples herein, including Compounds 1-32, ora pharmaceutically acceptable salt, solvate, and/or ester thereof, inthe preparation of a medicament for use in treating an Arenaviridaeinfection in a human.

VI. METHODS OF INHIBITION OF AN ARENA VIRIDAE POLYMERASE

Another aspect of the invention relates to methods of inhibiting theactivity of Arenaviridae polymerase comprising the step of treating asample suspected of containing Arenaviridae with a compound orcomposition of the invention.

Arenaviridae that can be treated using the methods of the presentinvention are single-stranded negative sense RNA viruses that typicallyinfect primates. Arenaviruses are able to multiply in virtually all celltypes.

Based upon studies in nonhuman primates infected with Lassa virus, thefirst cells infected appear to be dendritic cells in the lymphoidtissues. Infection progresses to infection of Kupffer cells in liver andparenchymal cells in liver and adrenal gland, endothelial cells in avariety of tissues including nervous tissue, and finally to infection ofthe epithelium. Evidence of liver infection in humans leading tohepatitis has also been documented) (Hensley, L., 2011, VirologyJournal; Yun, N. E., 2012 Viruses).

There are 30 identified genera of Arenaviruses: Allpahuayo virus (ALLY),Amapari virus (AMAV), Bear Canyon virus (BCNV), Catarina virus, Chaparevirus, Cupixi virus (CPXV), Dandenong virus, Flexal virus (FLEV),Guanarito virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodokovirus, Lassa virus (LASV; six strains—Josiah, NL, z148, Macenta, AV, andCSF), Latino virus (LATV), Lymphocytic choriomeningitis virus (LCMV),Lujo virus, Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus,Mopeia virus (MOPV), Oliveros virus (OLVV), Parana virus (PARV),Pichinde virus (PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus(SABV), Skinner Tank virus, Tacaribe virus (TCRV), Tamiami virus (TAMV),or Whitewater Arroyo virus (WWAV).

The arenavirus virions are heterogeneous in size from 40 to more than200 nm in diameter that consist of nucleocapsid surrounded by a lipidenvelope. Electron micrographs of the interior of virions show acharacteristic granular appearance due to incorporation of host cellribosomes in virus particles during assembly. The genome of arenavirusesconsists of two single-stranded RNA segments, small (S) and large (L).Both genomic segments have an ambisense gene organization and encode twogenes in opposite orientation. The L RNA (˜7 kb) encodes the viralRNA-dependent RNA polymerase (L) and the small RING finger zinc-bindingprotein (Z). The S RNA (˜3.4 kb) encodes the glycoprotein precursorprotein (GPC) and the nucleoprotein (NP). GPC is posttranslationallycleaved to yield two envelope glycoproteins GP1 and GP2 and the stablesignal peptide (SSP) (Yun, N. E., 2012 Viruses).

Compositions of the invention may act as inhibitors of arenaviruspolymerase, as intermediates for such inhibitors or have other utilitiesas described below. The inhibitors will bind to locations on the surfaceor in a cavity of Arenaviridae polymerase having a geometry unique toArenaviridae polymerase. Compositions binding Arenaviridae polymerasemay bind with varying degrees of reversibility. Those compounds bindingsubstantially irreversibly are ideal candidates for use in this methodof the invention. Once labeled, the substantially irreversibly bindingcompositions are useful as probes for the detection of Arenaviridaepolymerase. Accordingly, the invention relates to methods of detectingArenaviridae polymerase in a sample suspected of containing Arenaviridaepolymerase comprising the steps of: treating a sample suspected ofcontaining Arenaviridae polymerase with a composition comprising acompound of the invention bound to a label; and observing the effect ofthe sample on the activity of the label. Suitable labels are well knownin the diagnostics field and include stable free radicals, fluorophores,radioisotopes, enzymes, chemiluminescent groups and chromogens. Thecompounds herein are labeled in conventional fashion using functionalgroups such as hydroxyl, carboxyl, sulfhydryl or amino.

Within the context of the invention, samples suspected of containingArenaviridae polymerase include natural or man-made materials such asliving organisms; tissue or cell cultures; biological samples such asbiological material samples (blood, serum, urine, cerebrospinal fluid,tears, sputum, saliva, tissue samples, and the like); laboratorysamples; food, water, or air samples; bioproduct samples such asextracts of cells, particularly recombinant cells synthesizing a desiredglycoprotein; and the like. Typically the sample will be suspected ofcontaining an organism which produces Arenaviridae polymerase,frequently a pathogenic organism such as an Arenaviridae virus. Samplescan be contained in any medium including water and organic solvent\watermixtures. Samples include living organisms such as humans, and manmadematerials such as cell cultures.

The treating step of the invention comprises adding the composition ofthe invention to the sample or it comprises adding a precursor of thecomposition to the sample. The addition step comprises any method ofadministration as described above.

If desired, the activity of Arenaviridae polymerase after application ofthe composition can be observed by any method including direct andindirect methods of detecting Arenaviridae polymerase activity.Quantitative, qualitative, and semiquantitative methods of determiningArenaviridae polymerase activity are all contemplated. Typically one ofthe screening methods described above are applied, however, any othermethod such as observation of the physiological properties of a livingorganism are also applicable.

Organisms that contain Arenaviridae polymerase include the Arenaviridaevirus. The compounds of this invention are useful in the treatment orprophylaxis of Arenaviridae infections in animals or in man.

However, in screening compounds capable of inhibiting human Arenaviridaeviruses, it should be kept in mind that the results of enzyme assays maynot correlate with cell culture assays. Thus, a cell based assay shouldbe the primary screening tool.

In another embodiment, the present application provides for methods oftreating Arenaviridae virus infection in a human, comprising:administering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,solvate, and/or ester thereof. In some embodiments, the Arenaviridaeinfection is caused by an Arenaviridae virus. In some embodiments, theArenaviridae infection is caused by a Junin virus. In some embodiments,the Arenaviridae infection is caused by Lassa virus strains Josiah, N L,z148, Macenta, A V, or CSF. In some embodiments, an Arenaviridaepolymerase is inhibited.

The compounds of the present invention can be used in the treatment of ahuman already suffering from an Arenaviridae infection, or can beadministered prophylactically to reduce or prevent the chance of anArenaviridae infection. Physical examination of patients infected witharenavirus after the onset of fever often reveals purulent pharyngitis,bilateral conjunctival hemorrhages, facial edema, and generalizedabdominal tenderness. Macroscopic pathological changes can includepleural effusions, pulmonary edema, ascites, and hemorrhagicmanifestations in the gastrointestinal mucosa. Mortality rates forhospitalized patients vary between 5-10%.

VII. SCREENS FOR ARENA VIRIDAE POLYMERASE INHIBITORS

Compositions of the invention are screened for inhibitory activityagainst Arenaviridae polymerase by any of the conventional techniquesfor evaluating enzyme activity. Within the context of the invention,typically compositions are first screened for inhibition of Arenaviridaepolymerase in vitro and compositions showing inhibitory activity arethen screened for activity in vivo. Compositions having in vitro Ki(inhibitory constants) of less than about 5×10⁻⁶ M and preferably lessthan about 1×10⁻⁷ M are preferred for in vivo use.

Useful in vitro screens have been described in detail and will not beelaborated here. However, the examples describe suitable in vitroassays.

VIII. PREPARATION OF COMPOUNDS

The compounds of the present invention can be prepared by a variety ofmeans. For example, protected nucleosides of Formula V can be preparedby reaction of a protected lactone with an iodo-substituted base undersuitable coupling conditions. The nucleosides can then be modified witha prodrug moiety by reaction of a partially protected nucleoside with asuitable prodrug moiety, following be removal of the protecting groups,to afford the compounds of the present invention.

A. Preparation of Nucleosides via Iodo-Base

In some embodiments, the present invention provides a method ofpreparing a compound of Formula V:

The method of making the compound of Formula V includes forming areaction mixture having a coupling agent, a halo-silane, a compound ofFormula VI:

and a compound of Formula VII:

under conditions suitable to prepare the compound of Formula V, whereineach PG is independently a hydroxy protecting group, alternatively, twoPG groups on adjacent carbons can be combined to form a —C(R¹⁹)₂— group,R¹⁰ is H or a silyl group, and R¹⁹ is H, C₁-C₈ alkyl, phenyl orsubstituted phenyl.

Any suitable coupling agent can be used in the method of making thecompound of Formula V. The coupling agent can be a lithium couplingagent, a sodium coupling agent, a magnesium coupling agent, or others.For example, the coupling agent can be a deprotonating agent such asn-butyl lithium (n-BuLi), sodium hydride (NaH), lithium aluminum hydride(LAH or LiAlH₄), and others. The coupling agent can also be a magnesiumbased coupling agent such as, but not limited to, MgCl₂, iPrMgCl,tBuMgCl, PhMgCl, or combinations thereof. In some embodiments, thecoupling agent can be a lithium coupling agent or a magnesium couplingagent. In some embodiments, the coupling agent can be n-BuLi, MgCl₂,iPrMgCl, tBuMgCl, PhMgCl, or combinations thereof. In some embodiments,the coupling agent can be n-BuLi. In some embodiments, the couplingagent can be PhMgCl and iPrMgCl.

The coupling agent can be present in any suitable amount. For example,the coupling agent can be present in an amount of at least 1.0 eq.(mol/mol) to the compound of Formula V, such as about 1.0, 2, 3, 4, 5,6, 7, 8, 9, or about 10.0 eq. (mol/mol). The coupling agent can also bepresent in an amount of from about 1.0 to about 10.0 eq. (mol/mol) tothe compound of Formula V, such as of from about 1.0 to about 5.0 eq.(mol/mol), or of from about 1.0 to about 2.0 eq. (mol/mol). In someembodiments, the coupling agent can be present in an amount of fromabout 1.0 to about 5.0 eq. (mol/mol) to the compound of Formula V. Insome embodiments, the coupling agent can be present in an amount of fromabout 1.0 to about 2.0 eq. (mol/mol) to the compound of Formula V.

Any suitable halo-silane can be used in the method of making thecompound of Formula V. For example, the halo-silane can be afluoro-silane, a chloro-silane, a bromo-silane or an iodo-silane. Thesilane portion can have any suitable substituents, such as alkyl,alkenyl, alkynyl, cycloalkyl, or phenyl. Exemplary halo-silanes include,but are not limited to, Cl—Si(CH₃)₃, or Cl—Si(CH₃)₂CH₂CH₂Si(CH₃)₂—Cl. Insome embodiments, the halo-silane can be a chloro-silane. In someembodiments, the halo-silane can be Cl—Si(CH₃)₃, orCl—Si(CH₃)₂CH₂CH₂Si(CH₃)₂—Cl. In some embodiments, the halo-silane canbe TMS-Cl.

The silyl group of R^(m) can be any suitable group, but can depend onthe choice of the halo-silane. For example, when the halo-silane isTMS-Cl, the silyl group can be trimethylsilyl.

The halo-silane can be present in any suitable amount. For example, thehalo-silane can be present in an amount of at least 1.0 eq. (mol/mol) tothe compound of Formula V, such as about 1.0, 2, 3, 4, 5, 6, 7, 8, 9, orabout 10.0 eq. (mol/mol). The halo-silane can also be present in anamount of from about 1.0 to about 10.0 eq. (mol/mol) to the compound ofFormula V, such as of from about 1.0 to about 5.0 eq. (mol/mol), or offrom about 1.0 to about 2.0 eq. (mol/mol). In some embodiments, thehalo-silane can be present in an amount of from about 1.0 to about 5.0eq. (mol/mol) to the compound of Formula V. In some embodiments, thehalo-silane can be present in an amount of from about 1.0 to about 2.0eq. (mol/mol) to the compound of Formula V.

The hydroxy protecting group can be any protecting group suitable for ahydroxy functional group. Representative hydroxy protecting groupsinclude, but are not limited to, silanes such as trimethyl silane (TMS),t-butyl dimethyl silane (TBDMS), or t-butyl diphenyl silane (TBDPS),ethers such as methyl-methoxy (MOM), tetrahydropyran (THP), t-butyl,allyl, or benzyl, and esters such as acetyl, pivaloyl, or benzoyl. Insome embodiments, the hydroxy protecting group can be trimethyl silane(TMS), t-butyl dimethyl silane (TBDMS), t-butyl diphenyl silane (TBDPS),methyl-methoxy (MOM), tetrahydropyran (THP), t-butyl, allyl, benzyl,acetyl, pivaloyl, or benzoyl. In some embodiments, the hydroxyprotecting group can be benzyl.

Hydroxy groups on adjacent carbons, referred to as 1,2-hydroxy groups,can form a cyclic protecting group called an acetonide by reaction witha ketone of di-ether. Exemplary acetonides include, but are not limitedto acetonide and benzylidene acetal. In some embodiments, the hydroxyprotecting groups of hydroxy groups on adjacent carbons can be combinedto form acetonide.

When the R¹⁹ group is C₁-C₈ alkyl, R¹⁹ can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-buty, t-butyl, pentyl, iso-pentyl,neo-pentyl, hexyl, isohexyl, neohexyl, septyl or octyl. In someembodiments, the R¹⁹ group can be methyl.

Any suitable solvent can be used in the method of the present invention.Representative solvents include, but are not limited to, pentane,pentanes, hexane, hexanes, heptane, heptanes, petroleum ether,cyclopentanes, cyclohexanes, benzene, toluene, xylene,trifluoromethylbenzene, halobenzenes such as chlorobenzene,fluorobenzene, dichlorobenzene and difluorobenzene, methylene chloride,chloroform, acetone, ethyl acetate, diethyl ether, tetrahydrofuran, orcombinations thereof. In some embodiments, the solvent can betetrahydrofuran. Further representative solvents include, but are notlimited to 2-Methyltetrahydrofuran, Dibutyl ether, Methyl tert-butylether, Dimethoxyethane, Dioxanes (1.4 dioxane), N-methyl pyrrolidinone(NMP), or combinations thereof.

The reaction mixture of the method can be at any suitable temperature.For example, the temperature of the reaction mixture can be of fromabout −78° C. to about 100° C., or of from about −50° C. to about 100°C., or of from about −25° C. to about 50° C., or of from about −10° C.to about 25° C., or of from about 0° C. to about 20° C. In someembodiments, the temperature of the reaction mixture can be of fromabout 0° C. to about 20° C. In some embodiments, the temperature of thereaction mixture can be of from about −30° C. to about-10° C.

The reaction mixture of the method can be at any suitable pressure. Forexample, the reaction mixture can be at atmospheric pressure. Thereaction mixture can be also be exposed to any suitable environment,such as atmospheric gasses, or inert gasses such as nitrogen or argon.

The method of the present invention can provide the compound of FormulaV in any suitable yield. For example, the compound of Formula V can beprepared in a yield of at least about 50%, 55, 60, 65, 70, 75, 80, 85,90 or at least about 95%.

The method of the present invention can provide the compound of FormulaV in any suitable purity. For example, the compound of Formula V can beprepared in a purity of at least about 90, 95, 96, 97, 98 or at leastabout 99%. In some embodiments, the compound of Formula V can beprepared in at least 95% purity. In some embodiments, the compound ofFormula V can be prepared in at least 98% purity. In some embodiments,the compound of Formula V can be prepared in at least 99% purity.

In some embodiments, the method including preparing the compound ofFormula V:

wherein the method includes forming the reaction mixture having TMS-Cl,PhMgCl, iPrMgCl, the compound of Formula VI:

and the compound of Formula VII:

under conditions suitable to prepare the compound of Formula V.

In some embodiments, the present invention provides the compound:

B. Addition of Prodrug Moiety

The present invention also provides a method of coupling a prodrugmoiety to a nucleoside to provide a compound of the present invention.In some embodiments, the present invention provides a method ofpreparing a compound of Formula VIII:

wherein the method includes forming a reaction mixture including acoupling agent, a non-nucleophilic base, a compound of Formula IX:

and a compound of Formula X:

under conditions suitable to form the compound of Formula VIII, whereineach R^(x) is H or PG, each PG group is a hydroxy protecting group, orboth PG groups are combined to form —C(R¹⁹)₂—, R^(e1) and R^(e2) areeach independently H, C₁-C₆ alkyl or benzyl, R^(f) is H, C₁-C₈ alkyl,benzyl, C₃-C₆ cycloalkyl, or CH₂—C₃-C₆ cycloalkyl, 10⁹ is H, C₁-C₈alkyl, phenyl or substituted phenyl, and LG is a leaving group.

Any suitable coupling agent can be used in the method of making thecompound of Formula VIII, as described above for the method of makingthe compound of Formula V. In some embodiments, the coupling agent canbe a magnesium coupling agent. In some embodiments, the coupling agentcan be MgCl₂, iPrMgCl, tBuMgCl, PhMgCl, or combinations thereof. In someembodiments, the coupling agent can be MgCl₂.

Any suitable non-nucleophilic base can be used in the method of makingthe compound of Formula VIII. Representative non-nucleophilic basesinclude, but are not limited to, triethylamine, diisopropylethyl amine,N,N-diethylaniline, pyridine, 2,6-lutidine, 2,4,6-collidine,4-dimethylaminopyridine, and quinuclidine. In some embodiments, thenon-nucleophilic base can be di-isopropyl ethyl amine (DIPEA).

The protecting groups PG can be any suitable hydroxy protecting groups,as described above for the method of making the compound of Formula V.Exemplary protecting groups PG can be benzyl, or the PG groups can becombined to form an acetonide. Exemplary acetonides include, but are notlimited to acetonide and benzylidene acetal. In some embodiments, thehydroxy protecting groups of hydroxy groups on adjacent carbons can becombined to form acetonide. In some embodiments, the PG groups arecombined to form —C(R¹⁹)₂—. In some embodiments, each R^(a) is theprotecting group PG where the PG groups are combined to form —C(Me)₂—.

When the R^(e) group is C₁-C₈ alkyl, each Re can be methyl, ethyl,propyl, isopropyl, butyl, iso-butyl, sec-buty, t-butyl, pentyl,iso-pentyl, neo-pentyl, hexyl, isohexyl, neohexyl, septyl or octyl. Insome embodiments, each Re group can be methyl.

When the R^(f) group is C₁-C₈ alkyl, R^(f) can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-buty, t-butyl, pentyl, iso-pentyl,neo-pentyl, hexyl, isohexyl, neohexyl, septyl or octyl. In someembodiments, the R^(f) group can be methyl, ethyl, isopropyl, t-butyl,or iso-hexyl. When the R^(f) group is C₃-C₆ cycloalkyl, R^(f) can becyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments,R^(f) can be cyclobutyl, cyclopentyl or cyclohexyl.

When the R¹⁹ group is C₁-C₈ alkyl, R¹⁹ can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-buty, t-butyl, pentyl, iso-pentyl,neo-pentyl, hexyl, isohexyl, neohexyl, septyl or octyl. In someembodiments, the R¹⁹ group can be methyl.

The leaving group can be any suitable leaving group. Suitable leavinggroups LG include, but are not limited to, chloride, bromide, mesylate,tosylate, triflate, 4-nitrobenzenesulfonate, 4-chlorobenzenesulfonate,4-nitrophenoxy, pentafluorophenoxy, etc. In some embodiments, theleaving group LG can be 4-nitrophenoxy or pentafluorophenoxy. In someembodiments, the leaving group LG can be 4-nitrophenoxy.

In some embodiments, each R^(a) is PG where the PG groups are combinedto form —C(R¹⁹)₂—, R^(f) is C₁-C₈ alkyl, R¹⁹ is C₁-C₈ alkyl, and theleaving group LG is 4-nitrophenoxy or pentafluorophenoxy.

In some embodiments, the coupling agent is MgCl₂, and thenon-nucleophilic base is di-isopropyl ethyl amine.

In some embodiments, the compound of Formula VIII can be

In some embodiments, the compound of Formula VIII can be

In some embodiments, the compound of Formula VIII can be

In some embodiments, the method of making the compound Formula VIIIincludes forming the reaction mixture including MgCl₂, DIPEA, thecompound of Formula IX:

and the compound of Formula X:

under conditions suitable to form the compound of Formula VIII:

When the R^(a) groups of the compound of Formula VIII are the hydroxyprotecting groups PG, the method can include the additional step ofremoving the protecting groups to form the compound of Formula VIIIwhere each R^(a) is H. In some embodiments, the method of preparing thecompound of Formula VIII includes forming a second reaction mixtureincluding a deprotection agent and the compound Formula VIII whereineach R^(x) group is the protecting group PG, under suitable conditionsto form the compound of Formula VIII where each R^(x) is H. Thedeprotection agent can be any suitable agent to remove the protectinggroups PG such as hydrogen and a hydrogenation catalyst, or acid. Forexample, if the protecting group PG is benzyl, the deprotection agentcan be hydrogen and platinum on carbon. Alternatively, when theprotecting group PG is an acetonide, the deprotection agent can be anacid. Representative acids include, but are not limited to, acetic acid,glacial acetic acid, trifluoroacetic acid (TFA), hydrochloric acid,concentrated hydrochloric acid, and others. In some embodiments, themethod of preparing the compound of Formula VIII includes forming asecond reaction mixture including an acid and the compound Formula VIIIwherein the R^(a) groups are combined to form —C(R¹⁹)₂—, under suitableconditions to form the compound of Formula VIII where each R^(a) is H.In some embodiments, the acid can be hydrochloric acid.

Any suitable solvent can be used in the method of the present invention.Representative solvents include, but are not limited to, pentane,pentanes, hexane, hexanes, heptane, heptanes, petroleum ether,cyclopentanes, cyclohexanes, benzene, toluene, xylene,trifluoromethylbenzene, halobenzenes such as chlorobenzene,fluorobenzene, dichlorobenzene and difluorobenzene, methylene chloride,chloroform, acetone, ethyl acetate, diethyl ether, tetrahydrofuran,acetonitrile, or combinations thereof. In some embodiments, the solventcan be acetonitrile.

The reaction mixture of the method can be at any suitable temperature.For example, the temperature of the reaction mixture can be of fromabout −78° C. to about 100° C., or of from about −50° C. to about 100°C., or of from about −25° C. to about 50° C., or of from about −10° C.to about 25° C., or of from about 0° C. to about 20° C. In someembodiments, the temperature of the reaction mixture can be of fromabout 0° C. to about 20° C.

The reaction mixture of the method can be at any suitable pressure. Forexample, the reaction mixture can be at atmospheric pressure. Thereaction mixture can be also be exposed to any suitable environment,such as atmospheric gasses, or inert gasses such as nitrogen or argon.

The method of the present invention can provide the compound of FormulaVIII in any suitable yield. For example, the compound of Formula VIIIcan be prepared in a yield of at least about 50%, 55, 60, 65, 70, 75,80, 85, 90 or at least about 95%.

The method of the present invention can provide the compound of FormulaVIII in any suitable purity. For example, the compound of Formula VIIIcan be prepared in a purity of at least about 90, 95, 96, 97, 98 or atleast about 99%. In some embodiments, the compound of Formula VIII canbe prepared in at least 95% purity. In some embodiments, the compound ofFormula VIII can be prepared in at least 98% purity. In someembodiments, the compound of Formula VIII can be prepared in at least99% purity.

In some embodiments, the present invention provides the compound

IX. EXAMPLES

Certain abbreviations and acronyms are used in describing theexperimental details. Although most of these would be understood by oneskilled in the art, Table 1 contains a list of many of theseabbreviations and acronyms.

TABLE 1 List of abbreviations and acronyms. Abbreviation Meaning Ac₂Oacetic anhydride AIBN 2,2′-azobis(2-methylpropionitrile) Bn benzyl BnBrbenzylbromide BSA bis(trimethylsilyl)acetamide BzCl benzoyl chloride CDIcarbonyl diimidazole DABCO 1,4-diazabicyclo[2.2.2]octane DBN1,5-diazabicyclo[4.3.0]non-5-ene DDQ2,3-dichloro-5,6-dicyano-1,4-benzoquinone DBU1,5-diazabicyclo[5.4.0]undec-5-ene DCA dichloroacetamide DCCdicyclohexylcarbodiimide DCM dichloromethane DMAP4-dimethylaminopyridine DME 1,2-dimethoxyethane DMTCl dimethoxytritylchloride DMSO dimethylsulfoxide DMTr 4,4′-dimethoxytrityl DMFdimethylformamide EtOAc ethyl acetate ESI electrospray ionization HMDShexamethyldisilazane HPLC High pressure liquid chromatography LDAlithium diisopropylamide LRMS low resolution mass spectrum MCPBAmeta-chloroperbenzoic acid MeCN acetonitrile MeOH methanol MMTC monomethoxytrityl chloride m/z or m/e mass to charge ratio MH⁺ mass plus 1MH⁻ mass minus 1 MsOH methanesulfonic acid MS or ms mass spectrum NBSN-bromosuccinimide Ph phenyl rt or r.t. room temperature TBAFtetrabutylammonium fluoride TMSCl chlorotrimethylsilane TMSBrbromotrimethylsilane TMSI iodotrimethylsilane TMSOTf(trimethylsilyl)trifluoromethylsulfonate TEA triethylamine TBAtributylamine TBAP tributylammonium pyrophosphate TBSClt-butyldimethylsilyl chloride TEAB triethylammonium bicarbonate TFAtrifluoroacetic acid TLC or tlc thin layer chromatography Trtriphenylmethyl Tol 4-methylbenzoyl Turbo Grignard 1:1 mixture ofisopropylmagnesium chloride and lithium chloride δ parts per milliondown field from tetramethylsilane

A. Preparation of Compounds

Example 1 (2S)-ethyl 2-(chloro(phenoxy)phosphorylamino)propanoate(Chloridate A)

Ethyl alanine ester hydrochloride salt (1.69 g, 11 mmol) was dissolvedin anhydrous CH₂Cl₂ (10 mL) and the mixture stirred with cooling to 0°C. under N₂(g). Phenyl dichlorophosphate (1.49 mL, 10 mmol) was addedfollowed by dropwise addition of Et₃N over 10 min. The reaction mixturewas then slowly warmed to RT and stirred for 12 h. Anhydrous Et₂O (50mL) was added and the mixture stirred for 30 min. The solid that formedwas removed by filtration, and the filtrate concentrated under reducedpressure. The residue was subjected to silica gel chromatography elutingwith 0-50% EtOAc in hexanes to provide intermediate A (1.13 g, 39%). ¹HNMR (300 MHz, CDCl₃) δ 7.39-7.27 (m, 5H), 4.27 (m, 3H), 1.52 (m, 3H),1.32 (m, 3H). ³¹P NMR (121.4 MHz, CDCl₃) δ 8.2, 7.8.

Example 2 (2S)-2-ethylbutyl 2-(chloro(phenoxy)phosphorylamino)propanoate(Chloridate B)

The 2-ethylbutyl alanine chlorophosphoramidate ester B was preparedusing the same procedure as chloridate A except substituting2-ethylbutyl alanine ester for ethyl alanine ester. The material is usedcrude in the next reaction. Treatment with methanol or ethanol forms thedisplaced product with the requisite LCMS signal.

Example 3 (2S)-isopropyl 2-(chloro(phenoxy)phosphorylamino)propanoate(Chloridate C)

The isopropyl alanine chlorophosphoramidate ester C was prepared usingthe same procedure as chloridate A except substituting isopropyl alanineester for the ethyl alanine ester. The material is used crude in thenext reaction. Treatment with methanol or ethanol forms the displacedproduct with the requisite LCMS signal.

Example 4 (2R, 3R, 4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile(Compound 1)

The preparation of (2R, 3R, 4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrileis described below.

The commercially available lactol (10 g, 23.8 mmol) was dissolved inanhydrous DMSO (30 mL) under N₂(g). Ac2O (20 mL) was added and theresultant reaction mixture stirred at RT for 48 h. The reaction mixturewas poured onto ice H₂O (500 mL) and the mixture stirred for 20 min. Themixture was extracted with EtOAc (3×200 mL) and the combined organicextracts were then washed with H₂O (3×200 mL). The organic extract wasdried over anhydrous MgSO₄, filtered and concentrated under reducedpressure. The residue was dissolved in CH₂Cl₂ and subjected to silicagel chromatography eluting with 25% EtOAc in hexanes to provide thelactone (9.55 g, 96%). ¹H NMR (400 MHz, DMSO) δ 7.30-7.34 (m, 13H),7.19-7.21 (m, 2H), 4.55-4.72 (m, 6H), 4.47 (s, 2H), 4.28 (d, J=3.9 Hz,1H), 3.66 (m, 2H). LCMS m/z 436.1 [M+H₂O], 435.2 [M+OH]− Tr=2.82 min.HPLC Tr=4.59 [2-98% ACN in H2) over 5 min @2 ml/min flow.

The bromopyrazole (prepared according to WO2009/132135) (0.5 g, 2.4mmol) was suspended in anhydrous THF (10 mL) under N₂(g). The suspensionwas stirred and TMSCl (0.67 mL, 5.28 mmol) was added. The mixture wasstirred for 20 min. at RT and then cooled to −78° C. after which time asolution of n-BuLi (6 mL, 1.6N in hexanes, 9.6 mmol) was added slowly.The reaction mixture was stirred for 10 min. at −78° C. and then thelactone (1 g, 2.4 mmol) was added via syringe. When the reaction wascomplete as measured by LCMS, AcOH was added to quench the reaction. Themixture was concentrated under reduced pressure and the residuedissolved in a mixture of CH₂Cl₂ and H₂O (100 mL, 1:1). The organiclayer was separated and washed with H₂O (50 mL). The organic layer wasthen dried over anhydrous MgSO₄, filtered and concentrated under reducedpressure. The residue was subjected to silica gel chromatography elutingwith 0-50% EtOAc in hexanes to provide the product as a 1:1 mixture ofanomers (345 mg, 26% yield). LCMS m/z 553 [M+H].

The hydroxy nucleoside (1.1 g, 2.0 mmol) was dissolved in anhydrousCH₂Cl₂ (40 mL) and the solution cooled with stirring to 0° C. underN₂(g). TMSCN (0.931 mL, 7 mmol) was added and the mixture stirred for afurther 10 min. TMSOTf (1.63 mL, 9.0 mmol) was slowly added to thereaction and the mixture stirred for 1 h. The reaction mixture was thendiluted with CH₂Cl₂ (120 mL) and aqueous NaHCO₃ (120 mL) was added toquench the reaction. The reaction mixture was stirred for a further 10min and the organic layer separated. The aqueous layer was extractedwith CH₂Cl₂ (150 mL) and the combined organic extracts dried overanhydrous MgSO₄, filtered and concentrated under reduced pressure. Theresidue was dissolved in a minimal amount of CH₂Cl₂ and subjected tosilica gel chromatography eluting with a gradient of 0-75% EtOAc andhexanes to provide the tribenzyl cyano nucleoside as a mixture ofanomers. (0.9 g, 80%). ¹H NMR (300 MHz, CD₃CN) δ 7.94 (s, 0.5H), 7.88(s, 0.5H), 7.29-7.43 (m, 13H), 7.11-7.19 (m, 1H), 6.82-6.88 (m,1 H),6.70-6.76 (m, 1H), 6.41 (bs, 2H), 5.10 (d, J=3.9 Hz, 0.5H), 4.96 (d,J=5.1 Hz, 0.5H), 4.31-4.85 (m, 7H), 4.09-4.18 (m, 2H), 3.61-3.90 (m,2H). LCMS m/z 562 [M+H].

The tribenzyl cyano nucleoside (70 mg, 0.124 mmol) was dissolved inanhydrous CH₂Cl₂ (2 mL) and cooled to −78° C. under N₂(g). A solution ofBCl₃ (1N in CH₂Cl_(2, 0.506) mL, 0.506 mmol) was added and the reactionmixture stirred for 1 h. at −78° C. When the reaction was complete byLC/MS, MeOH was added to quench the reaction. The reaction mixture wasallowed to warm to room RT and the solvent removed under reducedpressure. The residue was subjected to C18 reverse phase HPLC, elutingfor 5 min with H₂O (0.1% TFA), followed by a gradient of 0-70% MeCN inH₂O (0.1% TFA) over 35 min, to elute the α-anomer (20 mg, 37%), andβ-anomer 1 (20 mg, 37%). (α-anomer) ¹H NMR (300 MHz, D₂O) δ 7.96 (s,1H), 7.20 (d, J=4.8 Hz, 1H), 6.91 (d, J=4.8 Hz, 1H), 4.97 (d, J=4.4 Hz,1H), 4.56-4.62 (m, 1H), 4.08-4.14 (m, 1H), 3.90 (dd, J=12.9, 2.4 Hz,1H), 3.70 (dd, J=13.2, 4.5 Hz, 1H). (β-anomer) ¹H NMR (400 MHz, DMSO) δ7.91 (s, 1H), 7.80-8.00 (br s, 2H), 6.85-6.89 (m, 2H), 6.07 (d, J=6.0Hz, 1H), 5.17 (br s, 1H), 4.90 (br s, 1H), 4.63 (t, J=3.9 Hz, 1H),4.02-4.06 (m, 1H), 3.94 (br s, 1H), 3.48-3.64 (m, 2H). LCMS m/z 292.2[M+H], 290.0 [M−H]. Tr=0.35 min. 13C NMR (400 MHZ, DMSO), 156.0, 148.3,124.3, 117.8, 117.0, 111.2, 101.3, 85.8, 79.0, 74.7, 70.5, 61.4. HPLCTr=1.32 min

Example 5(2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile(Compound 2)

The preparation of(2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrileis described below.

2-Deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose.1′-Methoxy-2-deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose (1.0 g, 2.88mmol) in TFA (13.5 mL) was treated with H₂O (1.5 mL) and the resultantmixture stirred for 5 h. The mixture was then diluted with EtOAc (100mL) and treated with saturated NaHCO₃ (50 mL). The organic layer wasseparated and washed with NaCl (50 mL), dried over anhydrous MgSO₄,filtered and concentrated under reduced pressure. The residue wassubjected to silica gel chromatography (80 g SiO₂Combiflash HP GoldColumn) eluting with 0-100% EtOAc in hexanes to afford2-deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose (695 mg, 72%) as a whitesolid: R_(f)=0.52 (25% EtOAc in hexanes). ¹H NMR (300 MHz, CDCl₃) δ 7.30(m, 10H), 5.35 (m, 1H), 4.68-4.29 (m, 7H), 3.70 (d, J=10.5 Hz, 1H), 3.50(d, J=10.5 Hz, 2H). ¹⁹F NMR (282.2 MHz, CDCl₃) δ −207 (m), −211 (m).LCMS m/z 350 [M+H₂O].

(3R, 4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one.2-Deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose (4.3 g, 12.8 mmol) wasdissolved in CH₂Cl₂ (85 mL) was treated with 4 ∪ MS (10 g) andpyridinium dichromate (14.4 g, 38.3 mmol). The resultant mixture wasstirred for 24 h and then filtered through a pad of Celite. The eluantwas concentrated under reduced pressure and the residue subjected tosilica gel chromatography (120 g SiO₂HP Gold Combiflash Column) elutingwith 0-100% EtOAc in hexanes to afford (3R, 4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one asa clear oil (3.5 g, 83%): R_(f)=0.25 (25% EtOAc in hexanes). ¹H NMR (300MHz, CDCl₃) δ 7.37 (m, 10H), 5.45 (dd, J=49,5.7, Hz, 1H), 4.85 (d,J=11.7 Hz, 1H), 4.52 (m, 4 H), 4.29 (d, J=5.4 Hz, 1H), 2.08 (dd, J=15.3,10.2 Hz, 2H). ¹⁹F NMR (282.2 MHz, CDCl₃) δ −216. LCMS m/z 348 [M+H₂O].HPLC (6-98% MeCNH₂O gradient, 0.05% TFA modifier) t_(R)=5.29 min.Phenomenex Synergi 4 m Hydro-RP 80 A, 50×4.60 mm, 4 micron; 2 mL/minflow rate

(3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-ol.7-Bromopyrrolo[1,2-f][1,2,4]-triazin-4-amine (68 mg, 0.319 mmol) in THF(1.4 mL) was treated with TMSCl (89 μL, 0.703 mmol) and the mixturestirred for 2 h. The mixture was then cooled to −78° C. and treated withnBuLi (1.0 M in hexanes, 1.09 mL, 1.09 mmol). The solution was stirredfor 30 min and then treated with (3R, 4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one(106 mg, 0.319 mmol) dropwise in THF (1.4 mL). The resultant mixture wasstirred for 30 min and then AcOH (83 μL, 1.44 mmol) in THF (1.0 mL) wasadded to quench the reaction. The mixture was warmed to RT and thenconcentrated under reduced pressure. The residue was diluted with EtOAc(100 mL) and washed with saturated NaCl solution (50 mL). The organiclayer was dried over anhydrous MgSO₄, filtered and concentrated underreduced pressure. The residue was subjected to silica gel chromatography(40 g SiO₂HP Gold Combiflash Column) eluting with 0-100% EtOAc inhexanes followed by a 0-100% gradient of (20% MeOH in EtOAc) in EtOAc toafford (3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-olas a white solid (68 mg, 44%, 60/40 mixture of α/β isomers). R_(f)=0.32(EtOAc). ¹H NMR (300 MHz, CDCl₃) δ 8.05 (s, 1H), 7.86 (s, 1H), 7.81 (s,1H), 7.64 (s, 1H), 7.26 (m, 10H), 6.95 (m, 1H), 6.71 (m, 1H), 6.08 (m,1H), 5.34 (m, 1H), 4.65 (m, 6H), 4.71 (m, 2H). ¹⁹F NMR (282.2 MHz,CDCl₃) δ −211 (m). LCMS m/z 465 [M+H]. HPLC (6-98% MeCNH₂O gradient,0.05% TFA modifier) t_(R)=4.37 min. (a-isomer), 4.54 min. ((3-isomer).

(3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carbonitrile:(3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-ol(195 mg, 0.42 mmol) was dissolved in MeCN (1.4 mL) was treated withTMSCN (336 μL, 2.52 mmol) and In(OTf)₃ (708 mg, 1.26 mmol). The solutionwas stirred at 70° C. for 18 h and then cooled to 0° C. The mixture wastreated with saturated NaHCO₃ solution (20 drops) then warmed to RT anddiluted with EtOAc (100 mL) and H₂O (50 mL). The organic layer wasseparated and washed with saturated NaCl solution (50 mL), dried overMgSO₄, filtered and concentrated under reduced pressure. The residue wassubjected to silica gel chromatography (40 g SiO₂HP Gold CombiflashColumn) eluting with 0-100% EtOAc in hexanes to afford (3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carbonitrileas a white solid (110 mg, 55%, 60/40 mixture of α/β isomers). Data forboth isomers: R_(f)=0.53 (EtOAc). ¹H NMR (300 MHz, CDCl₃) δ 8.01 (s,1H), 7.94 (s, 1H), 7.30 (m, 10H), 7.00 (d, J=4.5 Hz, 1H), 6.93 (d, J=4.8Hz, 1H), 6.87 (d, J=5.4 Hz, 1H), 6.70 (d, J=4.8 Hz, 1H), 5.85 (dd, J=52,3.3 Hz, 1H), 5.55 (dd, J=53, 4.5 Hz, 1H), 4.71 (m, 7H), 3.87 (m, 2H),3.72 (m, 2H). ¹⁹F NMR (282.2 MHz, CDCl₃) δ −196 (m), −203 (m). LCMS m/z474 [M+H]. HPLC (6-98% MeCNH₂O gradient, 0.05% TFA modifier) t_(R)=4.98min.

(2R, 3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (2) (3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carbonitrile(110 mg, 0.23 mmol) was dissolved in CH₂Cl₂ (1.5 mL) and cooled to 0° C.The reaction mixture was treated with BCl₃ (1.0 M in CH₂Cl_(2, 766) μL,0.77 mmol) and stirred for 2 h. The mixture was then cooled to −78° C.and treated with Et₃N (340 μL, 2.44 mmol) followed by MeOH (2 mL) beforeallowing to warm to RT. The reaction was concentrated under reducedpressure and then co-evaporated with MeOH (3×5 mL). The residue was thensuspended in H₂O (5 mL) and treated with NaHCO₃ (1 g). The solution wasstirred for 10 min and then concentrated under reduced pressure. Theresidue was filtered and washed with MeOH (3×10 mL) on a fritted glassfunnel (coarse) and the eluant concentrated under reduced pressure. Theresidue was subjected to reverse phase HPLC (6-98% MeCN in H₂O gradientwith 0.05% TFA modifier) to afford (2R, 3R, 4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile2 as a white solid (16.8 mg, 25%) and the a-isomer. Data for theI3-isomer: R_(f) =0.13 (10% MeOH in EtOAc). ¹H NMR (300 MHz, CD₃OD) δ8.09 (s, 1H), 7.28 (d, J=5.1 Hz, 1H), 7.17 (d, J=5.1 Hz, 1H), 5.42 (dd,J=53, 3.3 Hz, 1H), 4.20 (m, 2H), 3.99 (d, J=3.6 Hz, 1H), 3.77 (d, J=3.6Hz, 1H). ¹⁹F NMR (282.2 MHz, CDCl₃) δ −197 (m). LCMS m/z 294 [M+H]. HPLC(2-98% MeCNH₂O gradient, 0.05% TFA modifier) t_(R)=1.49 min.

Example 6 (2R, 3R, 4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)-5-methyltetrahydrofuran-3-ol(Compound 3)

The preparation of (2R, 3R, 4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)-5-methyltetrahydrofuran-3-olis described below.

The starting nucleoside (prepared as described in the synthesis ofcompound 2) (0.355 g, 0.765 mmol) was dissolved in anhydrous THF (35 mL)and cooled to 0° C. with stirring under N₂(g). A solution of methylmagnesium chloride (2 mL, 6 mmol) (3N in THF) was added and theresultant mixture stirred overnight. Acetic acid (7 mmol) was added toquench the reaction and then the solvents were removed by rotary underreduced pressure. The residue was re-dissolved in CH₂Cl₂ and thesolution subjected to a plug of silica gel to isolate the product (0.355g) as a crude mixture. LC/MS (m/z: 480, M⁺¹). The crude material wasdissolved in anhydrous CH₂Cl₂ (20 mL) and placed under N₂(g). Thesolution was stirred and treated with methanesulfonic acid (0.2 mL, 2.74mmol). The reaction mixture was stirred for 12 h at RT and then quenchedby the addition of Et₃N (3.5 mmol). The mixture was concentrated underreduced pressure and the residue subjected to silica gel chromatographyto provide the methyl substituted nucleoside (0.174 g, 0.377 mmol, 44%yield) as a 4:1 mixture of beta- and alpha-anomers respectively. ¹H NMR(300 MHz, CD₃CN) major anomer δ7.87 (s, 1H), 7.27-7.40 (m, 10 H), 6.77(d, J=4.5 HZ, 1H), 6.70 (d, J=4.5 Hz, 1H), 6.23 (br s, 2H), 5.53 (dd,J=55, 3.3 Hz, 1H), 4.42-4.75 (m, 4H), 4.19-4.26 (m, 1H), 3.65-4.00 (m,3H), 1.74 (d, J=3.9 Hz, 3H). ¹⁹F NMR (282.2 MHz, CD₃CN) major anomerδ−207 (m, 1F). LCMS m/z 463 [M+H].

The benzylated nucleoside material (0.134 g, 0.290 mmol), Degussacatalyst (0.268 g) and AcOH (30 mL) were mixed together. The reactionatmosphere was charged with H₂ (g) and the reaction stirred for 2 h. Thecatalyst was removed by filtration and the mixture concentrated underreduced pressure. The residue was dissolved in a minimal amount of H₂Oand subjected to reverse phase HPLC (C¹⁸ hydro RP column) to isolate theβ-anomer 3 (0.086 g, 0.217 mmol, 57% yield). ¹H NMR (300 MHz, D₂O) δ7.87 (s, 1H), 7.22 (d, J=4.8 Hz, 1H), 6.87 (d, J=4.8 Hz, 1H), 5.35 (dd,J=54, 3.6 Hz, 1H), 3.97-4.10 (m, 2H), 3.81 (dd, J=12.6, 2.1 Hz, 1H),3.64 (dd, J=12.6, 4.8 Hz, 1H), 1.65 (d, J=4.2 Hz, 3H). ¹⁹F NMR (282.2MHz, CD₃CN) δ −207 (m, 1F).

A small amount of alpha anomer was characterized as follows. ¹H NMR (300MHz, D₂O) δ 7.86 (s, 1H), 7.26 (d, J=4.8 Hz, 1H), 6.85 (d, J=4.8 Hz,1H), 5.31 (dd, J=54, 3.9 Hz, 1H), 4.39 (ddd, J=26.1, 9.9, 3.6 Hz, 2H),4.00-4.05 (m, 1H), 3.90 (dd, J=12.3, 2.1 Hz, 1H), 3.66 (dd, J=12.6, 4.8,1H), 1.56 (s, 3H). ¹⁹F NMR (282.2 MHz, CD₃CN) δ −198 (dd, J=54, 26 Hz,1F).

Example 7 (2R)-isopropyl 2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f[]1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphorylamino)propanoate(Compound 4)

The nucleoside 3 (0.011 g, 0.04 mmol) was dissolved intrimethylphosphate (2 mL) and cooled to 0° C. The mixture was stirredunder an atmosphere of N₂(g) and 1-Methylimidazole(0.320 mL, 5 mmol)followed by the alaninylmonoisopropyl, monophenol phosphorchloridate C(0.240 mL, 4.4 mmol) was added. The reaction mixture was stirred for 2h. at 0° C. and then allowed to warm slowly to RT. while monitoring byLC/MS. When complete by LCMS, the reaction mixture was treated with H₂O(5 mL) and then concentrated under reduced pressure. The residue wasdissolved in CH₂Cl₂ and subjected to silica gel chromatography elutingwith 0-100% EtOAc in hexanes. The product fractions were collected andconcentrated. The residue was subjected to prep HPLC to yield thealanine isopropyl monoamidate prodrug 4 as a mixture of isomers (4.7 mg,0.003 mmol, 6%). ¹H NMR (300 MHz, CD3CN) δ 7.87 (s, 1H), 7.17-7.44 (m, 5H), 6.71-6.83 (m, 2H), 6.14 (br, s, 2H), 5.38 (dd, J=56, 3.3 Hz, 1H),4.92-5.01 (m, 1H), 3.86-4.46 (m, 6H), 3.58 (m, 1H), 1.73 (m, 3H),1.18-1.34 (m, 9H). LCMS m/z 552 [M+H].

Example 8 (2R)-ethyl2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate(Compound 5)

The nucleoside 3 (0.026 g, 0.092 mmol) was dissolved intrimethylphosphate (2 mL) and cooled to 0° C. The mixture was stirredunder N₂(g) and 1-methylimidazole (0.062 mL, 0.763 mmol) followed by thechloridate A (0.160 g, 0.552 mmol) were added. The reaction mixture wasstirred for 2 h. at 0° C. and then allowed to warm slowly to RT. H₂O (5mL) was added to quench the reaction and then the mixture concentratedunder reduced pressure. The residue was dissolved in CH₂Cl₂ andsubjected to silica gel chromatography eluting with 0-100% EtOAc inhexanes. The product fractions were collected and concentrated. Crudeproduct was eluted using 0 to 100 percent EtOAc in hexanes. The crudeproduct was collected and concentrated under reduced pressure. Theresidue was subjected to prep HPLC to yield 5 (2.0 mg, 4% yield). LCMSm/z 538 [M+H].

Example 9 ((2R, 3R, 4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (Compound 6)

The nucleoside 3 (0.022 g, 0.056 mmol) was dissolved intrimethylphosphate (1 mL) and stirred under N₂(g). Phosphorousoxychloride (0.067 mL, 0.73 mmol) was added and the mixture stirred for2 h. Monitoring by analytical ion-exchange column determined the time atwhich >80 percent of monophosphate was formed. A solution oftributylamine (0.44 mL, 1.85 mmol) and triethylammonium pyrophosphate(0.327 g, 0.72 mmol) dissolved in anhydrous DMF (1 mL) was added. Thereaction mixture was stirred for 20 min and then quenched by theaddition of 1N triethylammonium bicarbonate solution in H₂O (5 mL). Themixture was concentrated under reduced pressure and the residuere-dissolved in H₂O. The solution was subjected to ion exchangechromatography to yield the title product 6 (1.7 mg, 6% yield). LCMS m/z521 [M−H]. Tr=0.41. HPLC ion exchange TR=9.40 min

Example 10(2R,3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carbonitrile(Compound 7)

The preparation of(2R,3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carbonitrileis described below.

((3α,R,5S,6αR)-2,2-dimethyl-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methanol.The acetate material (1.2 g, 5.5 mmol) (J. Org. Chem. 1985, 50, 3547, DeBernardo et al) was dissolved in a 1:1 mixture MeOH and THF (10 mL). A1N solution of NaOH(aq) (10 mL) was added until the pH was 13. Thereaction mixture was stirred for 2 h and then neutralized to pH 8-9 bythe addition of AcOH. The mixture was extracted with EtOAc (10×30 mL)and the combined organic extracts dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure. The residue was subjected tosilica gel chromatography eluting with 0-70% EtOAc in hexanes to givethe desired product (866 mg, 90%). ¹H NMR (300 MHz, CDCl₃) δ 5.84 (d,J=3.6 Hz, 1H), 4.78 (t, J=4.5 Hz, 1H), 4.38 (m, 1H), 3.93-3.54 (m, 2H),2.04-1.84 (m, 2H), 1.52 (s, 3H), 1.33 (s, 3H).

(3αR,5S,6α,R)-5-(benzyloxymethyl)-2,2-dimethyl-tetrahydrofuro[2,3-d][1,3]dioxole.Sodium hydride (188 mg, 7.46 mmol) was dissolved in anhydrous THF (5 mL)and stirred under N₂(g) at RT. The alcohol (866 mg, 4.97 mmol) wasdissolved in anhydrous THF (3 mL) and then added in portions over 5 min.to the sodium hydride mixture. The resultant mixture was stirred for 20min. and then benzyl bromide (892 μL, 7.46 mmol) was added. The reactionwas stirred for 2 h and then poured onto a mixture of ice cold aqueousNaHCO₃ and EtOAc (30 mL). The organic layer was separated and then theaqueous layer re-extracted with EtOAc (30 mL). The combined organicextracts were dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was subjected to silica gelchromatography eluting with 0-40% EtOAc in hexanes to give the benzylether product (912 mg, 69%). ¹H NMR (300 MHz, CDCl₃) δ 7.35-7.27 (m,5H), 5.86 (d, J=3.6 Hz, 1H), 4.74 (t, J=4.2 Hz, 1H), 4.60 (s, 2H), 4.42(m, 1H), 3.69-3.53 (m, 2H), 2.10-2.04 (m, 1H), 1.83-1.77 (m, 1H), 1.52(s, 3H), 1.33 (s, 3H).

(3R,5S)-5-(benzyloxymethyl)-tetrahydrofuran-2,3-diol. The benzyl ether(910 mg, 3.44 mmol) was dissolved in a 1:1 AcOH and H₂O (20 mL) mixtureand stirred at 60° C. for 7 h. The mixture was concentrated underreduced pressure and the residue subjected to silica gel chromatographyeluting with 0-70% EtOAc in hexanes to give the diol product (705 mg,91%). ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.27 (m, 5H), 5.40 (d, J=3.9 Hz,0.5H), 5.17 (s, 0.5H), 4.67-4.56 (m, 3H), 4.33 (m, 0.5H), 4.24 (d, J=4.8Hz, 0.5H), 3.71-3.67 (m, 1H), 3.56-3.42 (m, 2H), 2.31-2.22 (m, 1H),2.08-1.89 (m, 2H).

(3R,5S)-5-(benzyloxymethyl)-3-hydroxy-dihydrofuran-2(3H)-one. The diol(705 mg, 3.14 mmol) was dissolved in benzene (30 mL) and treated with asilver carbonate celite mixture (3.46 g, 6.28 mmol). The resultantmixture was stirred at 80° C. under N₂(g) for 2 h. The mixture was thencooled to RT, filtered and concentrated under reduced pressure. Theresidue was subjected to silica gel chromatography eluting with 0-70%EtOAc in hexanes to give the lactone product (600 mg, 86%). ¹H NMR (300MHz, CDCl₃) δ 7.39-7.27 (m, 5H), 4.75-4.68 (m, 1H), 4.60-4.49 (m, 2H),3.74-3.54 (m, 2H), 2.61-2.35 (m, 2H), 2.38-2.28 (m, 1H).

(3R, 5S)-3-(benzyloxy)-5-(benzyloxymethyl)-dihydrofuran-2(3H)-one. Thelactone (600 mg, 2.7 mmol) was dissolved in EtOAc (30 mL) and treatedwith silver oxide (626 mg, 2.7 mmol) followed by benzyl bromide (387 μL,3.24 mmol). The reaction mixture was then stirred at 50° C. under N₂(g)for 8 h. Additional silver oxide (300 mg) was then added and theresultant mixture stirred at 50° C. for 16 h. Additional benzyl bromide(50 uL) and silver oxide (150 mg) were added and the mixture stirred foran additional 8 h. The reaction mixture was allowed to cool, filteredand then concentrated under reduced pressure. The residue was subjectedto silica gel chromatography eluting with 0-20% EtOAc in hexanes to givethe title product (742 mg, 88%). ¹H NMR (300 MHz, CDCl₃) δ 7.39-7.27 (m,10H), 4.99 (d, J=11.4 Hz, 1H), 4.72 (m, 2H), 4.56 (m, 2H), 4.39 (t,J=8.1 Hz, 1H), 3.72-3.51 (m, 2H), 2.42-2.25 (m, 2H).

(3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-(benzyloxy)-5-(benzyloxymethyl)-tetrahydrofuran-2-ol.The 7-bromopyrrolo[1,2-f][1,2,4]triazin-4-amine (607 mg, 2.85 mmol) wasdissolved in anhydrous THF (10 mL) and stirred under Ar(g) at RT. TMSCl(1.1 mL, 8.55 mmol) was added dropwise and the mixture stirred for 2 h.The reaction was concentrated under reduced pressure and then driedunder high vacuum. The residue was suspended in THF (20 mL) and stirredunder Ar(g) at −78° C. A 2.5M n-BuLi solution in hexane (2.28 mL, 5.7mmol) was added dropwise over 10 min. and the resultant mixture stirredfor 60 min. The lactone (742 mg, 2.37 mmol) dissolved in anhydrous THF(7 mL) was added to the above mixture over 20 min. The reaction mixturewas stirred for 2 h. and then quenched with AcOH until pH was 5-6. Themixture was allowed to warm to RT and then diluted with EtOAc. Thesolution was washed with saturated NaHCO₃ solution, saturated NaCl,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was subjected to silica gel chromatography eluting with 0-80%EtOAc in hexanes to give the title product (250 mg, 24%). LCMS m/z 447.2[M+H], 445.1 [M−H].

(3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-(benzyloxy)-5-(benzyloxymethyl)-tetrahydrofuran-2-carbonitrile.The alcohol (250 mg, 0.56 mmol) was dissolved in anhydrous CH₂Cl₂(10 mL)and stirred under Ar(g) at −15° C. TMSCN (448 μL, 3.36 mmol) was addeddropwise and the mixture stirred for 10 min. TMSOTf (466 μL, 2.58 mmol)was added dropwise over 10 min and the resultant mixture stirred for 90min. at −15° C. Additional TMSCN (224 μL, 3 eq.) and TMSOTf (202 μL, 2eq.) was added and stirring continued for 5 h. Saturated aqueous NaHCO₃solution was added to quench the reaction and the mixture stirred for 10min. The organic layer was separated and washed with saturated aqueousNaHCO₃ solution, saturated NaCl solution, dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. The residue wassubjected to silica gel chromatography eluting with 0-70% EtOAc inhexanes to give the title product (150 mg, 59%). LCMS m/z 456.3 [M+H],454.1 [M−H].

(2R,3R,5S)₂(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carbonitrile(7). The benzyl ether (150 mg, 0.329 mmol) was dissolved in anhydrousCH₂Cl₂ (2 mL) and the mixture stirred under Ar(g) at −20° C. A 1M BCl₃solution in CH₂Cl₂ (724 μL, 0.724 mmol) was added dropwise and theresultant mixture stirred for 2 h. Additional 1M BCl₃ in CH₂Cl₂ (724 μL,0.724 mmol) was added and stirring continued for 2 h. The mixture wasthen cooled to −78° C. and slowly treated with a 2:1 mixture of Et₃N andMeOH (3 mL). The mixture was stirred for 10 min and then treated withMeOH (10 mL). The reaction was allowed to warm to RT and thenconcentrated under reduced pressure. The residue was dissolved in MeOHand concentrated under reduced pressure. The residue was dissolved inMeOH again and treated with solid NaHCO₃. The mixture was stirred for 5min and then the solid removed by filtration. The solution wasconcentrated under reduced pressure and subjected to preparative HPLC toprovide the desired product 7 (10 mg, 11%). ¹H NMR (300 MHz, D₂O) δ 7.71(s, 1H), 6.75 (d, J=4.5 Hz, 1H), 6.65 (d, J=4.8 Hz, 1H), 4.91 (t, J=6.3Hz, 1H), 4.57 (m, 1H), 3.67-3.47 (m, 2H), 2.18 (m, 2H). LCMS m/z 276.1[M+H], 274.0 [M−H].

Example 11 (2S)-isopropyl2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(Phenoxy)-phosphorylamino)propanoate(Compound 8)

The nucleoside 1 (45 mg, 0.15 mmol) was dissolved in anhydrous trimethylphosphate (0.5 mL) and the solution stirred under N₂(g) at 0° C. Methylimidazole (36 μL, 0.45 mmol) was added to the solution.Chlorophosphoramidate C (69 mg, 0.225 mmol) was dissolved in anhydrousTHF (0.25 mL) and added dropwise to the nucleoside mixture. When thereaction was complete by LCMS, the reaction mixture was diluted withEtOAc and washed with saturated aqueous NaHCO₃ solution, saturated NaCl,dried over anhydrous Na2SO₄, filtered and concentrated under reducedpressure. The residue was subjected to silica gel chromatography elutingwith 0-5% MeOH in CH₂Cl₂ followed by preparative HPLC to give theproduct (20.9 mg, 25%). ¹H NMR (300 MHz, CD₃OD) δ 7.95 (m, 1H),7.31-6.97 (m, 7H), 4.94 (m, 1H), 4.78 (m, 1H), 4.43 (m, 3H), 4.20 (m,1H), 3.80 (d, 1H), 1.30-1.18 (m, 9H). ³¹P NMR (121.4 MHz, CD₃OD) δ 3.8.LCMS m/z 561.0 [M+H], 559.0 [M−H].

Example 12

(2S)-2-ethylbutyl2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate(Compound 9)

Compound 9 can be prepared by several methods described below.

Procedure 1

Prepared from Compound 1 and chloridate B according to the same methodas for the preparation of compound 8. ¹H NMR (300 MHz, CD₃OD) δ 7.87 (m,1H), 7.31-7.16 (m, 5H), 6.92-6.89 (m, 2H), 4.78 (m, 1H), 4.50-3.80 (m,7H), 1.45-1.24 (m, 8H), 0.95-0.84 (m, 6H). ³¹P NMR (121.4 MHz, CD₃OD) δ3.7. LCMS m/z 603.1 [M+H], 601.0 [M−H].

Procedure 2

(2S)-2-ethylbutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate. (2S)-2-ethylbutyl 2#(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (1.08 g, 2.4 mmol)was dissolved in anhydrous DMF (9 mL) and stirred under a nitrogenatmosphere at RT.(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (350 mg, 1.2 mmol) wasadded to the reaction mixture in one portion. A solution oft-butylmagnesium chloride in THF (1M, 1.8 mL, 1.8 mmol) was then addedto the reaction dropwise over 10 minutes. The reaction was stirred for 2h, at which point the reaction mixture was diluted with ethyl acetate(50 mL) and washed with saturated aqueous sodium bicarbonate solution(3×15 mL) followed by saturated aqueous sodium chloride solution (15mL). The organic layer was dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The resulting oil was purified withsilica gel column chromatography (0-10% MeOH in DCM) to afford(2S)-2-ethylbutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (311 mg, 43%, 1:0.4 diastereomeric mixture at phosphorus) asa white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.85 (m, 1H), 7.34-7.23 (m,2H), 7.21-7.09 (m, 3H), 6.94-6.84 (m, 2H), 4.78 (d, J=5.4 Hz, 1H),4.46-4.33 (m, 2H), 4.33-4.24 (m, 1H), 4.18 (m, 1H), 4.05-3.80 (m, 3H),1.52-1.39 (m, 1H), 1.38-1.20 (m, 7H), 0.85 (m, 6H). ³¹P NMR (162 MHz,CD₃OD) δ 3.71, 3.65. LCMS m/z 603.1 [M+H], 600.9 [M−H]. HPLC (2-98%MeCN—H₂O gradient with 0.1% TFA modifier over 8.5 min, 1.5 mL/min,Column: Phenomenex Kinetex C18, 2.6 um 100 Å, 4.6×100 mm) t_(R)=5.544min, 5.601 min

Separation of the (S) and (R) Diastereomers

(2S)-2-ethylbutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate was dissolved in acetonitrile. The resulting solution wasloaded onto Lux Cellulose-2 chiral column, equilibrated in acetonitrile,and eluted with isocratic acetonitrile/methanol (95:5 vol/vol). Thefirst eluting diastereomer had a retention time of 17.4 min, and thesecond eluting diastereomer had a retention time of 25.0 min.

First Eluting Diastereomer is (S)-2-ethylbutyl2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate:

¹H NMR (400 MHz, CD₃OD) δ 8.05 (s, 1H), 7.36 (d, J=4.8 Hz, 1H), 7.29 (brt, J=7.8 Hz, 2H), 7.19-7.13 (m, 3H), 7.11 (d, J=4.8 Hz, 1H), 4.73 (d,J=5.2 Hz, 1H), 4.48-4.38 (m, 2H), 4.37-4.28 (m, 1H), 4.17 (t, J=5.6 Hz,1H), 4.08-3.94 (m, 2H), 3.94-3.80 (m, 1H), 1.48 (sep, J=12.0, 6.1 Hz,1H), 1.34 (p, J=7.3 Hz, 4H), 1.29 (d, J=7.2 Hz, 3H), 0.87 (t, J=7.4 Hz,6H). ³¹PNMR (162 MHz, CD₃OD) δ 3.71 (s). HPLC (2-98% MeCN—H₂O gradientwith 0.1% TFA modifier over 8.5 min, 1.5 mL/min, Column: PhenomenexKinetex C18, 2.6 um 100 Å, 4.6×100 mm) t_(R)=5.585 min.

Second Eluting Diastereomer is (S)-2-ethylbutyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate:

¹HNMR (400 MHz, CD₃OD) δ 8.08 (s, 1H), 7.36-7.28 (m, 3H), 7.23-7.14 (m,3H), 7.08 (d, J=4.8 Hz, 1H), 4.71 (d, J=5.3 Hz, 1H), 4.45-4.34 (m, 2H),4.32-4.24 (m, 1H), 4.14 (t, J=5.8 Hz, 1H), 4.08-3.94 (m, 2H), 3.93-3.85(m, 1H), 1.47 (sep, J=6.2 Hz, 1H), 1.38-1.26 (m, 7H), 0.87 (t, J=7.5 Hz,6H). ³¹PNMR (162 MHz, CD₃OD) δ 3.73 (s). HPLC (2-98% MeCN—H₂O gradientwith 0.1% TFA modifier over 8.5 min, 1.5 mL/min, Column: PhenomenexKinetex C18, 2.6 um 100 Å, 4.6×100 mm) t_(R)=5.629 min.

Example 13 (2S)-ethyl2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate(Compound 10)

The preparation of (2S)-ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Procedure 1. Preparation via Chloridate A

Prepared from Compound 1 and chloridate A using same method as for thepreparation of compound 8. ¹H NMR (300 MHz, CD₃OD) δ 7.95 (m, 1H),7.32-6.97 (m, 7H), 4.78 (m, 1H), 4.43-4.08 (m, 6H), 3.83 (m, 1H),1.31-1.18 (m, 6H). ³¹P NMR (121.4 MHz, CD₃OD) δ 3.7. LCMS m/z 547.0[M+H], 545.0 [M−H].

Procedure 2. Preparation via Nitro-Benzene Compound L

Compound 1 (50 mg, 0.17 mmol) was dissolved in NMP-THF (1:1 mL)) andcooled with ice bath. tBuMgCl (0.257 mL, 0.257 mmol) was then added over5 min. The resulting mixture was allowed to warm to RT and was stirredfor 30 min. Then a solution of compound L (Prepared according toUS20120009147, 74.6 mg, 0.189 mmol) in THF (2 mL) was added. After 30min, the reaction mixture was purified by HPLC (acetonitrile 10 to 80%in water) to give compound 29 as a yellow solid. The solid was furtherpurified with silica gel chromatography (MeOH 0 to 20% DCM) to affordcompound 29 (23 mg, 24% as a 2.5:1 mixture of diastereomers). ¹H NMR(400 MHz, CD₃OD) δ 7.76 (d, J=6.0 Hz, 1H), 7.25-7.14 (m, 2H), 7.11-6.99(m, 3H), 6.87-6.72 (m, 2H), 4.70 (d, J=5.4 Hz, 1H), 4.39-4.24 (m, 2H),4.20 (dddd, J=9.7, 7.9, 5.1, 2.8 Hz, 1H), 4.10 (dt, J=12.8, 5.5 Hz, 1H),4.06-3.91 (m, 2H), 3.72 (ddq, J=14.3, 9.3, 7.1 Hz, 1H), 1.17 (dd, J=7.1,1.0 Hz, 1H), 1.14-1.06 (m, 5H). ³¹P NMR (162 MHz, CD₃OD) δ 3.73, 3.68.MS m/z=547 (M+1)⁺.

Example 14 (2S)-ethyl2-((((2R,3R,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate(Compound 11)

Compound 11 was prepared from Compound 2 and chloridate A using samemethod as for the preparation of compound 8. ¹H NMR (300 MHz, CD₃OD) δ7.91 (m, 1H), 7.33-7.16 (m, 5H), 6.98-6.90 (m, 2H), 5.59 (m, 1H),4.50-4.15 (m, 4H), 4.12-3.90 (m, 3H), 1.33-1.18 (m, 6H). ³¹P NMR (121.4MHz, CD₃OD) δ 3.8. LCMS m/z 549.0 [M+H], 547.1 [M−H].

Example 15 (2S,2′S)-diethyl2,2′-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate(Compound 12)

The nucleoside 1 (14.6 mg, 0.05 mmol) was dissolved in anhydroustrimethyl phosphate (0.5 mL) and stirred under N₂(g) at RT. POCl₃ (9.2μL, 0.1 mmol) was added and the mixture stirred for 60 min. Alanineethyl ester hydrochloride (61 mg, 0.4 mmol) and then Et₃N (70 μL, 0.5mmol) was added. The resultant mixture was stirred for 15 min. and thenadditional Et₃N (70 μl, 0.5 mmol) was added to give a solution pH of9-10. The mixture was stirred for 2 h. and then diluted with EtOAc,washed with saturated aqueous NaHCO₃ solution followed by saturatedaqueous NaCl solution. The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was subjected topreparative HPLC (C₁₈ column) to yield the product 12 (5.5 mg, 16%). ¹HNMR (400 MHz, CD₃OD) δ 8.13 (s, 1H), 7.41 (d, J=4.8 Hz, 1H), 7.18 (d,J=4.8 Hz, 1H), 4.78 (d, J=5.6 Hz, 1H), 4.36 (m, 1H), 4.25-4.08 (m, 7H),3.83 (m, 2H), 1.33-1.23 (m, 12H). ³¹P NMR (121.4 MHz, CD₃OD) δ 13.8.LCMS m/z 570.0 [M+H], 568.0 [M−H].

Example 16(2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethynyl-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(Compound 13)

The preparation of(2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethynyl-5-(hydroxymethyl)tetrahydrofuran-3,4-diolis described below.

The nucleoside alcohol (0.6 g, 1.08 mmol) (prepared as described inCompound 1 synthesis) was dissolved in anhydrous THF (8 mL) and placedunder N₂(g). The reaction mixture was stirred and cooled to 0° C. andthen treated with a 0.5N solution of ethynyl magnesium bromide in THF(17.2 mL, 17.2 mmol). The reaction mixture was stirred overnight at RT.AcOH (1.5 mL) was added to quench the reaction. The mixture wasconcentrated under reduced pressure and the residue redissolved inCH₂Cl_(2.) The solution subjected to a plug of silca gel eluting with 0to 80% EtOAc in Hexanes to provide the title product as a crude mixture.LCMS m/z 579 [M+H].

The crude ethynyl alcohol (0.624 g, 1.08 mmol) was dissolved inanhydrous CH₂Cl₂ (10 mL) and placed under N₂(g). The mixture was stirredand sulfonic acid (0.2 mL, 2.74 mmol) was added. The reaction mixturewas stirred for 12 h. at RT. When complete by LCMS, Et₃N (0.56 mL) wasadded to quench the reaction. The reaction was concentrated underreduced pressure and the residue subjected to silica gel chromatographyeluting with 0 to 75% EtOAc in Hexanes to yield the ethynyl nucleosideas a mixture of anomers (0.200 g, 33% over 2 steps). LCMS m/z 561 [M+H].

The tribenzyl nucleoside (0.650 g, 1.16 mmol) was dissolved in anhydrousCH₂Cl₂ (30 mL) and cooled to −78° C. under N₂(g). A solution of borontribromide (1 N in CH₂Cl₂, 5.5 mL) was added and the reaction mixturestirred for 1 h. at −78° C. A solution of MeOH (10 mL) and pyridine (2mL) was added to quench the reaction and the mixture was allowed to riseto RT. The mixture was concentrated under reduced pressure and subjectedto preparative HPLC to provide the α-anomer (20 mg) and β-anomer 13 (110mg). (β-anomer) ¹H NMR (300 MHz, DMSO) δ 7.81 (s, 1H), 7.76 (br s, 2H),6.80-6.85 (m, 2H), 5.11 (d, J=7.2 Hz, 1H), 4.90 (d, J=6.0 Hz, 1H), 4.82(dd, J=7.2, 4.8 Hz, 1H), 4.62 (t, J=6.3 Hz, 1H), 3.95-3.99 (m, 1H),3.85-3.91 (dd, J=11.4, 5.7 Hz, 1H), 3.61-3.67 (m, 1H), 3.47-3.55 (m,1H), 3.52 (d, J=0.9 Hz, 1H). (α-anomer) ¹H NMR (300 MHz, DMSO) δ 7.80(s, 1H), 7.59 (bs, 2H), 6.80 (d, J=4.5 Hz, 1H), 6.54 (d, J=4.2 Hz, 1H),5.00 (d, J=7.2 Hz, 1H), 4.89 (d, J=4.8 Hz, 1H), 4.74 (t, J=5.7 Hz, 1H),4.58 (t, J=4.5 Hz, 1H), 4.27 (m, 1H), 3.88 (m, 1H), 3.64-3.72 (m, 1H),3.51-3.59 (m, 1H), 3.48 (d, J=0.6 Hz, 1H). LCMS m/z 291 [M+H].

Example 17(2R,3R,4R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-1,3,4-tris(benzyloxy)hexane-2,5-diol(Compound 14)

The preparation of(2R,3R,4R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-1,3,4-tris(benzyloxy)hexane-2,5-diolis described below.

The tribenzyl alcohol from Compound 1 synthesis (0.250 g, 0.453 mmol)was dissolved in anhydrous THF (25 mL) and stirred under N₂(g). Thereaction mixture was cooled to 0° C. and then a 3.0 N solution of methylmagnesium chloride in THF(1.2 mL, 3.62 mmol) was added. The reactionmixture was stirred overnight at RT. Acetic acid (1.5 mL) was added toquench the reaction and then the mixture was concentrated under reducedpressure. The residue was redissolved in CH₂Cl₂ and subjected to a plugof silca gel eluting with 0 to 80% EtOAc in hexanes. The crude product(0.452 g) was then used in the next reaction without furtherpurification. LCMS m/z 569 [M+H].

The crude methyl nucleoside (0.452 g, 0.796 mmol) was dissolved inanhydrous CH₂Cl₂ (20 mL) and stirred under N₂(g). Methanesulfonic acid(0.2 mL, 2.78 mmol) was added and the reaction stirred for 12 hr at RT.Et₃N (0.56 mL) was added to quench the reaction and then the mixtureconcentrated under reduced pressure. The residue was subjected to silicagel chromatography eluting with 0 to 75% EtOAc in Hexanes to yield theproduct as a mixture of anomers (0.20 g, 46% over 2 steps). LCMS m/z 551[M+H].

The tribenzyl nucleoside (0.20 g, 0.364 mmol) was dissolved in AcOH (30mL). and charged with Pd/C (Degussa) (400 mg). The stirred mixture wasflushed with N₂(g) three times and then H₂ (g) was introduced, Thereaction was stirred under H₂ (g) for 2 h. and then the catalyst removedby filtration. The solution was concentrated under reduced pressure andunder the residue was re-dissolved in H₂O. The solution was subjected topreparative HPLC under neutral conditions to provide the α-anomer andβ-anomer 14 in 81% yield. (α-anomer) ¹H NMR (300 MHz, D₂O) δ 7.81 (s,1H), 7.22 (d, 1H), 6.75 (d, 1H), 4.47 (d, 1H), 4.25-4.31 (m, 1H),3.88-4.95 (m, 1H), 3.58-3.86 (dd, 2H), 1.50 (s, 3H). (β-anomer)¹H NMR(300 MHz, D₂O) δ 7.91 (s, 1H), 7.26 (d, 1H), 6.90 (d, 1H), 4.61 (d, 1H),4.00-4.09 (m, 2H), 3.63-3.82 (dd, 2H), 1.67 (s, 3H). LCMS m/z 281 [M+H].

Example 18S,S′-2,2′-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate) (Compound 15)

The nucleoside 1 (0.028 g, 0.096 mmol) was dissolved intrimethylphosphate (1 mL). The reaction was stirred under N₂(g) and thentreated with 1H-tetrazole (0.021 g, 0.29 mmol). The reaction mixture wascooled to 0° C. and the phosphane (Nucleoside Nucleotides, Nucleicacids; 14; 3-5; 1995; 763-766. Lefebvre, Isabelle; Pompon, Alain;Perigaud, Christian; Girardet, Jean-Luc; Gosselin, Gilles; et al.) (87mg, 0.192 mmol) was added. The reaction was stirred for 2 h. and thenquenched with 30% hydrogen peroxide (0.120 mL). The mixture was stirredfor 30 min at RT and then treated with saturated aqueous sodiumthiosulfate (1 mL). The mixture was stirred for 10 min. and thenconcentrated under reduced pressure. The residue was subjected topreparative HPLC to isolate the title product 15. ¹H NMR (300 MHz,CD₃CN) δ 7.98 (s, 1H), 6.92 (d, 1H), 6.81 (d, 1H), 6.44 (bs, 2H), 4.82(m, 2H), 4.47 (m, 1H), 4.24 (m, 2H), 4.00 (m, 4H), 3.80 (bs, 1H), 3.11(m, 4H), 1.24 (s, 9H). ³¹P NMR (121.4 MHz, CD₃CN) δ −1.85 (s). LCMS m/z661 [M+H].

Example 19 S,S′-2,2′-((((2R, 3S, 4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate) (Compound 16)

Compound 16 was prepared using the same method as compound 15 exceptsubstituting compound 13 as the starting nucleoside. ¹H NMR (300 MHz,CD₃CN) δ 7.91 (s, 1H), 6.86 (d, J=4,8 Hz, 1H), 6.76 (d, J=4.5 Hz, 1H),6.29 (bs, 2H), 4.69 (t, J=2.7 Hz, 1H), 4.58 (d, J=5.7 Hz, 1H), 4.14-4.33(m, 5H), 3.99-4.07 (m, 4H), 3.53 (d, J=5.4 Hz, 1H), 3.11 (q, J=5.7 Hz,4H), 1.22 (s, 18H). LCMS m/z 658.9 [M+]. Tr=2.31

Example 20 ((2R, 3S, 4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (Compound 17)

Compound 17 was prepared from compound 1 using a similar procedure tothe preparation of compound 6. The product was isolated as the sodiumsalt. ¹H NMR (400 MHz, D₂O) δ 7.76 (s, 1H), 6.88 (d, J=4.8 Hz, 1H), 6.73(d, J=4.4 Hz, 1H), 4.86 (d, J=5.2 Hz, 1H), 4.43 (m, 1H), 4.39 (m, 1H),4.05 (m, 1H), 3.94 (m, 1H). ³¹P NMR (121.4 MHz, D₂O) δ −5.4 (d, 1P),−10.8 (d, 1P), −21.1 (t, 1P). LCMS m/z 530 [M−H], 531.9 [M+H] Tr=0.22min. HPLC ion exchange Tr=9.95 min.

Example 21 ((2R, 3S, 4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (Compound 18)

Compound 18 was prepared from compound 13 using a similar procedure tothe preparation of compound 6. The product was isolated as the TEA salt.¹H NMR (300 MHz, D₂O) δ 7.85 (s, 1H), 7.09 (d, J=4.6 Hz, 1H), 6.95 (d,J=4.7 Hz, 1H), 4.23 (m, 2H), 4.08 (m, 2H), 3.06 (q, J=7.4 Hz, 20H), 1.14(t, J=7.3 Hz, 30H). ³¹P NMR (121.4 MHz, D₂O) δ −10.8 (d, 1P), −11.2 (d,1P), −23.2 (t, 1P). LCMS m/z 530.8 [M+H], Tr=0.46. HPLC ion exchangeTr=9.40 min.

Example 22 ((2R, 3S, 4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (Compound 19)

Compound 19 was prepared from compound 14 using a similar procedure tothe preparation of compound 6. ¹H NMR (400 MHz, D₂O) δ 7.78 (s, 1H),6.98 (m, 1H), 6.84 (m, 1H), 4.45 (m, 1H), 4.04 (m, 4H), 1.54 (s, 3H).³¹P NMR (161 MHz, D₂O) δ −10.6 (m), −23.0 (m). LCMS m/z 521.0 [M+H].

Example 23((2R,3R,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate (Compound 20)

Compound 20 was prepared from compound 2 using a similar procedure tothe preparation of compound 6. ¹1H NMR (400 MHz, D₂O) δ 7.78 (s, 1H),6.93 (d, J=4.4 Hz, 1H), 6.78 (d, J=4.8 Hz, 1H), 5.45 (dd, J=53, 4.4 Hz,1H), 4.38-4.50 (m, 2H), 4.13-4.20 (m, 2H). ³¹P NMR (161 MHz, D₂O) δ −5.7(d, 1P), −11.0 (d, 1P), −21.5 (t, 1P). LCMS m/z 533.9.0 [M+H], 532.0[M−H] Tr=1.25 min. HPLC ion exchange Tr=11.0 min.

Example 24 (2S)-ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate(21)

The preparation of (2S)-ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoateis described below.

Preparation of (S)-ethyl 2-amino-3-phenylpropanoate hydrochloride

L-Phenylalanine (5 g, 30 mmol) was taken up in EtOH (30 mL). TMSCl(6.915 mL, 54 mmol) was added to the reaction at RT. The reaction vesselwas fitted with a reflux condenser and the reaction was placed in an 80°C. bath. The reaction was stirred overnight. The next day the reactionwas cooled to RT, concentrated under reduced pressure and the resultingresidue was taken up in Et₂O. The resulting slurry was filtered and theisolate solids were further washed with Et₂O. The washed solids wereplaced under high vacuum to yield example (S)-ethyl2-amino-3-phenylpropanoate hydrochloride (6.86 g, 99%). ¹H NMR (400 MHz,DMSO-d₆) δ 8.52 (s, 3H), 7.30 (m, 5H), 4.24 (ABX, J_(AX)=7.8 Hz,J_(BX)=6.2 Hz, 1H), 4.11 (m, 2H), 3.17, 3.05 (ABX, J_(AB)=−14 Hz,J_(BX)=5.8 Hz, J_(AX)=7.6 Hz, 2H), 1.09 (t, J=6.8 Hz, 3H).

Preparation of (2S)-ethyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate(Compound D)

(S)-ethyl 2-amino-3-phenylpropanoate hydrochloride (1.01 g, 4.41 mmol)was dissolved in DCM (50 mL). This solution was cooled to 0° C. andPhOP(O)Cl₂ (0.656 mL, 4.41 mmol) was added, followed by the slowaddition of Et₃N (1.62 mL, 11.5 mmol) over 5 min. The cold bath wasremoved and the reaction was allowed to warm to RT and stir over aperiod of 80 min. p-NO₂PhOH (0.583 g, 4.19 mmol) was added, followed bymore Et₃N (0.3 mL, 2.1 mmol). The reaction progress was monitored byLC/MS. Upon completion of the reaction, it was diluted with Et₂O, andthe resulting solids were removed by filtration. The filtrate wasconcentrated and compound D (1.25 g, 60%, as a mixture of diastereomers)was isolated by silica gel column chromatography (25 g dry loadcartridge, 120 g column; eluent: 100% hexanes ramping to 55% EtOAc inhexanes). ¹H NMR (400 MHz, CD₃OD) δ 8.17 (m, 2H), 7.33 (m, 2H),7.09-7.25 (m, 10H), 4.17 (m, 1H), 4.07 (m, 2H), 3.08 (m, 1H), 2.84 (m,1H), 1.14 (m, 3H). ³¹P NMR (162 MHz, DMSO-d₆) δ −1.479 (s), −1.719 (s).MS m/z=471.01 [M+1].

Preparation of (2S)-ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate(Compound 21)

Compound 1 (0.030 g, 0.103 mmol) was dissolved in DMF (1 mL) and thenTHF (0.5 mL) was added. t-BuMgCl (1M/THF, 154.5 μL, 0.154 μmol) wasadded to the reaction in a drop-wise manner with vigorous stirring. Theresulting white slurry was stirred at RT for 30 min. A solution ofcompound D (0.058 g, 0.124 mmol) in THF (1 mL) was added in a drop-wisemanner to the reaction at RT. The reaction progress was monitored byLC/MS. When the reaction progressed to 50% conversion, the reaction wascooled in an ice bath and quenched with glacial acetic acid (70 μL). Thereaction was concentrated and compound 21 (22 mg, 34%, as a 2.6:1mixture of diastereomers) was isolated from the residue by reverse phaseHPLC. ¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J=4 Hz, 1H), 7.90 (brs, 2H),7.09-7.30 (m, 8H), 7.01, (t, J=8.2 Hz, 2H), 6.89 (d, J=4.4 Hz, 1H), 6.82(t, J=4.4 Hz, 1H), 6.27 (m, 1H), 6.14 (m, 1H), 5.34 (m, 1H), 4.62 (t,J=5.6 Hz, 1H), 4.15 (m, 1H), 3.78-4.01 (m, 6H), 2.92 (m, 1H), 2.78 (m,1H), 1.04 (m, 3H). ³¹P NMR (162 MHz, DMSO-d₆) δ 3.69 (s), 3.34 (s). MSm/z=623.0 [M+H].

Example 25 (2S)-ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryDamino)-3-methylbutanoate(22)

The preparation of (2S)-ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoateis described below.

Preparation of (2S)-ethyl3-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino) butanoate(Compound E)

The (S)-ethyl 2-amino-3-methylbutanoate (0.351 g, 1.932 mmol) wasdissolved in DCM (17 mL). This solution was cooled in an ice bath andPhOP(O)Cl₂ (0.287 mL, 1.932 mmol) was added, followed by the slowaddition of Et₃N (1.62 mL, 11.4 mmol) over 5 min. The cold bath wasremoved and the reaction was allowed to warm to RT and stir over aperiod of 1 h. p-NO₂PhOH (0.255 g, 1.836 mmol) was added, and thereaction progress was monitored by LC/MS. Upon completion of thereaction, the mixture was diluted with Et₂O, and the resulting solidswere removed by filtration. The filtrate was concentrated and compound E(0.642 g, 79% as a mixture of diastereomers) was isolated by silica gelcolumn chromatography (12 g dry load cartridge, 80 g column; eluent:100% hexanes ramping to 55% EtOAc in hexanes). ¹H NMR (400 MHz, DMSO-d₆)δ 8.30 (d, J=9.2 Hz, 2H), 7.48 (t, J=9.6 Hz, 2H), 7.40 (t, J=7.8 Hz,2H), 7.20-7.27 (m, 3H), 6.60 (quart, J=11.6 Hz, 1H), 4.01 (m, 2H), 3.61(m, 1H), 1.93 (m, 1H), 1.11 (m, 3H), 0.79 (m, 6H). ³¹P NMR (162 MHz,DMSO-d₆) δ −0.342 (s), −0.578 (s). MS m/z=422.9 [M+H].

Preparation of (2S)-ethyl 2-(((((2R,3 S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoate(Compound 22)

Compound 1 (0.040 g, 0.137 mmol) was dissolved in NMP (1.5 mL) and thenTHF (0.25 mL) was added. This solution was cooled in an ice bath andt-BuMgCl (1M/THF, 425.7 μL, 0.426 μmol) was added in a drop-wise mannerwith vigorous stirring. The ice bath was removed and the resulting whiteslurry was stirred at RT for 15 min. A solution of compound E (0.081 g,0.192 mmol) in THF (0.5 mL) was added in a drop-wise manner to thereaction at RT. The reaction progress was monitored by LC/MS. When thereaction progressed to 50% conversion, the reaction was cooled in an icebath and quenched with glacial acetic acid (70 μL). The reaction wasconcentrated and compound 22 (22 mg, 34%) was semi-purified from theresidue by reverse phase HPLC. The semi-pure material was furtherpurified by silica gel column chromatography (12 g dry load cartridge,40 g column; eluent: 100% EtOAc ramping to 10% MeOH in EtOAc) to yieldcompound 22 (0.034 g, 43% as a 1.8:1 mixture of diastereomers). ¹H NMR(400 MHz, DMSO-d₆) δ 7.91 (d, J=1.6 Hz, 1H), 7.88 (brs, 2H), 7.32 (m,2H), 7.15 (m, 3H), 6.90 (t, J=4.2 Hz, 1H), 6.84 (d, J=4.8 Hz, 1H), 6.26(dd, J=13.4, 6.2 Hz, 1H), 5.87 (quart. J=11.2 Hz, 1H), 5.35 (m, 1H),4.64 (m, 1H), 4.25 (m, 2H), 3.93-4.15 (m, 4H), 3.45 (m, 1H), 1.87 (m,1H), 1.09-1.16 (m, 3H), 0.70-0.83 (m,6 H). ³¹P NMR (162 MHz, DMSO-d₆) δ4.59 (s), 4.47 (s). MS m/z=575.02 [M+H].

Example 26 (S)-isopropyl2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(23)

The preparation of (S)-isopropyl2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Compound 1 (60.0 mg, 206 μmol) was dissolved in NMP (0.28 mL). THF (0.2mL) was added followed by tert-butyl magnesium chloride (1.0M solutionin tetrahydrofuran, 0.309 mL) at RT under an argon atmosphere. After 20min, a solution of compound F (Prepared according to Cho, A. et al J.Med. Chem. 2014, 57, 1812-1825., 81 mg, 206 μmol) in THF (0.2 mL) wasadded, and the resulting mixture was warmed to 50° C. After 3 h, thereaction mixture was allowed to cool to RT and was purified directly bypreparatory HPLC (Phenominex Synergi 4u Hydro-RR 80 Å 150×30 mm column,5-100% acetonitrile/water gradient) to afford compound 23 (44 mg, 38% asa single diastereomer). ¹H NMR (400 MHz, CD₃OD) δ 7.86 (s, 1H),7.34-7.26 (m, 2H), 7.21-7.12 (m, 3H), 6.91 (d, J=4.6 Hz, 1H), 6.87 (d,J=4.6 Hz, 1H), 4.92 (sept, J=6.3 Hz, 1H), 4.80 (d, J=5.4 Hz, 1H),4.43-4.34 (m, 1H), 4.33-4.24 (m, 1H), 4.18 (t, J=5.6 Hz, 1H), 3.82 (dq,J=9.7, 7.1 Hz, 2H), 1.27 (dd, J=7.1, 1.0 Hz, 3H), 1.18 (dd, J=6.3, 4.8Hz, 6H). ³¹P NMR (162 MHz, CD₃OD) δ 3.72 (s). t_(R)=1.39 min, MSm/z=561.11 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: ThermoLCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50×4.6 mm; Solvents: ACNwith 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0 min-2.0min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN,3.2 min-3.5 min 2% ACN at 2 μl/min. HPLC: t_(R)=2.523 min; HPLC system:Agilent 1100 series.; Column: Gemini 5μ C18 110A, 50×4.6 mm; Solvents:ACN with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98%ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

Example 27 (2S)-cyclobutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(24)

The preparation of (2S)-cyclobutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Preparation of (25)-cyclobutyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (Compound G)

Phenyl dichlorophosphate (1.49 mL, 10 mmol) was dissolved in 10 mL ofanhydrous DCM and stirred under atmosphere nitrogen in an ice bath.L-Alanine isobutyl ester hydrochloride (0.9 g, 5 mmol) was added in oneportion. Triethylamine (765 μL, 5.5 mmol) was then added dropwise.Reaction stirred for 1 h. More Triethylamine (765 μL, 5.5 mmol) wasadded dropwise and the reaction was stirred for 45 min. p-Nitrophenol(1.25 g, 9 mmol) was added in one portion and stirred for 30 min.Triethylamine (765 μL, 5.5 mmol) was added and the reaction mixture wasstirred for 2 h. Additional p-nitrophenol (1.25 g, 9 mmol) andtriethylamine (765 μL, 5.5 mmol) were then added, and the reaction wasstirred for another 2 h. The reaction mixture was concentrated underreduced pressure. The resulting crude was diluted with EtOAc and washedtwice with 5% aqueous citric acid solution, followed with saturatedaqueous sodium chloride solution. The organic layer was then dried overanhydrous sodium sulfate and concentrated under reduced pressure. Thecrude residue was purified with silica gel column (0-20-50% EtOAc inhexanes) to give compound G (1.48 g, 70% yield as a mixture ofdiastereomers). ¹H NMR (400 MHz, CD₃OD) δ 8.33-8.23 (m, 2H), 7.52-7.33(m, 4H), 7.33-7.17 (m, 3H), 4.96-4.85 (m, 1H), 4.07-3.96 (m, 1H), 2.27(m, 2H), 2.07-1.91 (m, 2H), 1.83-1.70 (m, 1H), 1.70-1.55 (m, 1H), 1.32(m, 3H). ³¹P NMR (162 MHz, CD₃OD) δ −1.36, −1.59. MS m/z=420.9 [M+H].

Preparation (2S)-cyclobutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(Compound 24)

Compound 1 (58 mg, 0.2 mmol) was mixed with compound G (101 mg, 0.24mmol) in 2 mL of anhydrous DMF. Magnesium chloride (42 mg, 0.44 mmol)was added in one portion. The reaction mixture was heated to 50° C.DIPEA (87 μL, 0.5 mmol) was added, and the reaction was stirred for 2 hat 50° C. The reaction mixture was cooled to room temperature, wasdiluted with EtOAc and was washed with 5% aqueous citric acid solutionfollowed by saturated aqueous sodium chloride solution. The organiclayer was then dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The crude residue was purified with silica gelcolumn (0-2-5% MeOH in DCM) to afford compound 24 (42 mg, 37% yield, asa mixture of diastereomers). ¹H NMR (400 MHz, Methanol-d4) δ 7.85 (m,1H), 7.34-7.22 (m, 2H), 7.22-7.08 (m, 3H), 6.94-6.84 (m, 2H), 4.95-4.85(m, 1H), 4.79 (m, 1H), 4.46-4.34 (m, 2H), 4.34-4.24 (m, 1H), 4.19 (m,1H), 3.81 (m, 1H), 2.27 (m, 2H), 2.01 (m, 2H), 1.84-1.68 (m, 1H), 1.62(m, 1H), 1.30-1.16 (m, 3H). ³¹P NMR (162 MHz, cd₃od) δ 3.70, 3.65. MSm/z=573.0 [M+H].

Example 28 (2S)-isopropyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate(25)

The preparation of (2S)-isopropyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoateis described below.

Preparation of (2S)-isopropyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate(Compound H)

Phenyl dichlorophosphate (718 μL, 4.8 mmol) was dissolved in 10 mL ofanhydrous DCM and stirred under a nitrogen atmosphere in an ice bath.L-Phenylalanine isopropyl ester hydrochloride (1 g, 4.1 mmol) was addedin one portion. Another 10 mL of anhydrous DCM was added. Triethylamine(736 μL, 5.3 mmol) was added dropwise and the reaction mixture wasstirred for 30 min. More triethylamine (736 μL, 5.3 mmol) was then addeddropwise and the reaction mixture was stirred for 30 min. Additionaltriethylamine (736 μL, 5.3 mmol) was then added dropwise and thereaction mixture was stirred for 15 min. p-Nitrophenol (600 mg, 4.32mmol) was then added. The ice bath was then removed and the reactionmixture was allowed to warm to room temperature and stirred for 2 h.More p-nitrophenol (50 mg) and triethylamine (736 μL, 5.3 mmol) were theadded and the reaction mixture was stirred for 1 h.

The reaction mixture was then concentrated under reduced pressure, andwas diluted with EtOAc and washed twice with 5% aqueous citric acidsolution, followed with saturated aqueous sodium chloride solution. Theorganic layer was dried over anhydrous sodium sulfate and wasconcentrated under reduced pressure. The crude was purified with silicagel column (0-15% EtOAc in hexanes) to give compound II (1.57 g, 68%yield as a mixture of diastereomers). ¹H NMR (400 MHz, CDCl₃) δ 8.17 (m,2H), 7.38-7.13 (m, 10H), 7.13-7.02 (m, 2H), 4.95 (m, 1H), 4.31 (m, 1H),3.69 (m, 1H), 3.02 (dd, J=6.1, 1.8 Hz, 2H), 1.21-1.08 (m, 6H). ³¹P NMR(162 MHz, cdcl3) δ −2.96, −2.98. MS m/z=485.0 [M+H].

Preparation of (2S)-isopropyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate(Compound 25)

Compound 1 (58 mg, 0.2 mmol) and compound H (116 mg, 0.24 mmol) weremixed and 2 mL of anhydrous D1VIF was added. The reaction mixture wasstirred under a nitrogen atmosphere at room temperature. 1M tBuMgCl inTHF (300 μL, 0.3 mmol) was added dropwise over 3 minutes and thereaction mixture was then stirred for 16 h. The reaction mixture wasdiluted with EtOAc and washed with 5% aqueous citric acid solution,saturated aqueous sodium bicarbonate solution and then saturated aqueoussodium chloride solution. The organic layer was dried over anhydroussodium sulfate and concentrated under reduced pressure. The cruderesidue was purified with silica gel column (0-5% MeOH in DCM) to givecompound 25 (40 mg, 32% yield as a mixture of diastereomers). ¹H NMR(400 MHz, CD₃OD) δ 7.84 (m, 1H), 7.27-7.08 (m, 8H), 7.08-6.97 (m, 2H),6.88 (m, 2H), 4.91-4.84 (m, 1H), 4.74 (m, 1H), 4.26 (m, 1H), 4.19-4.04(m, 2H), 4.04-3.91 (m, 2H), 2.97 (m, 1H), 2.82 (m, 1H), 1.14 (m, 3H),1.06 (m, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 3.63, 3.25. MS m/z=637.0 [M+H].

Example 29 (S)-methyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(26)

The preparation of (S)-methyl 2-(((S)-(((2R,3 S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooledwith an ice water bath. Then 1M t-BuMgCl (0.52 mL, 0.77 mmol) was addeddropwise slowly. The resulting mixture was stirred for 30 min at roomtemperature. Then compound I (Prepared according to WO 2012142085, 219mg, 0.52 mmol) in THF (2 mL) was added over 5 min and the resultingmixture was stirred for 24 h at room temperature. The reaction mixturewas then diluted with EtOAc, cooled under ice-water bath, washed with aqNaHCO₃ (2 mL), washed with brine, dried with sodium sulfate, andconcentrated in vacuo. The resulting mixture was purified by silica gelcolumn chromatography (MeOH 0 to 20% in DCM) and prep-HPLC (acetonitrile10 to 80% in water) to give compound 26 (12 mg, 6.6% as a singlediastereomer). 1H NMR (400 MHz, CD₃OD) δ 7.86 (s, 1H), 7.29 (dd, J=8.6,7.2 Hz, 2H), 7.21-7.09 (m, 3H), 6.94-6.81 (m, 2H), 4.79 (d, J=5.4 Hz,1H), 4.38 (ddq, J=10.8, 5.3, 2.7 Hz, 2H), 4.33-4.23 (m, 1H), 4.18 (t,J=5.5 Hz, 1H), 3.86 (dq, J=9.9, 7.1 Hz, 1H), 3.62 (s, 3H), 1.27 (dd,J=7.2, 1.1 Hz, 3H). MS m/z=533 (M+1)⁺.

Example 30 (S)-neopentvl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(27)

The preparation of (S)-neopentyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooledunder ice water bath. Then 1M t-BuMgCl (0.52 mL, 0.77 mmol) was addeddropwise slowly. The resulting mixture was stirred for 30 min at roomtemperature. Then compound J (Prepared according to WO2012075140, 248mg, 0.52 mmol) was added over 5 min and the resulting mixture wasstirred for 24 h at room temperature, diluted with EtOAc, cooled underice-water bath, treated with aq NaHCO₃ (2 mL), washed with brine, driedwith sodium sulfate, and concentrated in vacuo. The resulting mixturewas purified by silica gel column chromatography (MeOH 0 to 20% in DCM)and prep-HPLC (acetonitrile 10 to 80% in water) to give Compound 27 (12mg, 10% as a single diastereomer). ¹H NMR (400 MHz, CD₃OD) δ 7.86 (s,1H), 7.36-7.24 (m, 2H), 7.23-7.10 (m, 3H), 6.96-6.85 (m, 2H), 4.78 (d,J=5.4 Hz, 1H), 4.38 (tdd, J=10.0, 4.9, 2.5 Hz, 2H), 4.32-4.24 (m, 1H),4.17 (t, J=5.6 Hz, 1H), 3.91 (dq, J=9.8, 7.1 Hz, 1H), 3.81 (d, J=10.5Hz, 1H), 3.69 (d, J=10.5 Hz, 1H), 1.31 (dd, J=7.2, 1.1 Hz, 3H), 0.89 (s,9H). MS m/z=589 (M+1)⁺.

Example 31 (2S)-cyclopentvl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(28)

The preparation of (2S)-cyclopentyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooledunder ice water bath. Then 1M t-BuMgCl (0.52 mL, 0.77 mmol) was addeddropwise slowly. The resulting mixture was stirred for 30 min at roomtemperature. Then compound K (Prepared according to WO2012075140, 247mg, 0.52 mmol) in THF (2 mL) was added over 5 min and the resultingmixture was stirred for 24 h at room temperature, diluted with EtOAc,cooled under ice-water bath, treated with aq NaHCO₃ (2 mL), washed withbrine, dried with sodium sulfate, and concentrated in vacuo. Theresulting mixture was purified by silica gel column chromatography (MeOH0 to 20% in DCM) and prep-HPLC (acetonitrile 10 to 80% in water) to giveexample 28 (47 mg, 23% as a 27:1 mixture of diastereomers). 1H NMR (400MHz, CD₃OD) δ 7.85 (s, 1H), 7.33-7.22 (m, 2H), 7.14 (tdd, J=7.6, 2.1,1.1 Hz, 3H), 6.95-6.87 (m, 2H), 5.13-5.00 (m, 1H), 4.78 (d, J=5.4 Hz,1H), 4.48-4.35 (m, 2H), 4.30 (ddd, J=10.6, 5.7, 3.6 Hz, 1H), 4.19 (t,J=5.4 Hz, 1H), 3.78 (dq, J=9.2, 7.1 Hz, 1H), 1.81 (dtd, J=12.5, 5.9, 2.4Hz, 2H), 1.74-1.49 (m, 6H), 1.21 (dd, J=7.1, 1.2 Hz, 3H). MS m/z=587(M+1)⁺.

Example 32 (2S)-cyclohexyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(29)

The preparation of (2S)-cyclohexyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

To a mixture of compound 1 (50 mg, 0.343 mmol), compound M (Preparedaccording to US20130143835, 93 mg, 0.209 mmol), and MgCl₂ (24.5 mg,0.257 mmol) in DMF (1 mL) was added diisopropylethylamine (0.075 mL,0.43 mmol) dropwise over 5 min at 0° C. The resulting mixture wasstirred at 50° C. for 1 h. The reaction mixture was then cooled with anice-water bath, treated with 1M citric acid (0.5 mL), and was purifieddirectly by prep-HPLC (ACN 0 to 70% in water) to afford compound 29 (20mg, 19% as a mixture of diastereomers). ¹H NMR (400 MHz, CD₃OD) δ 7.84(s, 1H), 7.32-7.23 (m, 2H), 7.18-7.10 (m, 3H), 6.93-6.87 (m, 2H), 4.78(d, J=5.4 Hz, 1H), 4.67 (td, J=8.7, 4.2 Hz, 1H), 4.48-4.35 (m, 2H), 4.30(ddd, J=10.8, 5.7, 3.7 Hz, 1H), 4.20 (t, J=5.4 Hz, 1H), 3.88-3.71 (m,1H), 1.83-1.63 (m, 4H), 1.58-1.46 (m, 1H), 1.46-1.24 (m, 5H), 1.24 (s,3H). ³′P NMR (162 MHz, CD₃OD) δ 3.75. MS m/z=601 (M+1)⁺.

Example 33 Ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate(30)

The preparation of ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoateis described below.

Preparation of Ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate

Take up triphenylphosphine (6.18 g, 25.00 mmol) in THF (30 mL). Nextcharge DIAD (4.92 mL, 25.00 mmol) and stir at room temperature for 10min. Dissolve 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid(5.08 g, 25.00 mmol) in THF (20 mL) and add to the reaction mixturefollowed by the addition of ethanol (2.19 mL, 37.49 mmol). Allow thereaction to stir at room temperature for 1 h. The solvents were removedunder reduced pressure and the crude was taken up in 1:1 Et₂O:Hexanes(120 mL). The solid triphenylphosphine oxide was filtered off and thesolvent was removed under reduced pressure. The crude was taken up inminimal CH₂Cl₂ and purified by silica gel chromatography 0-50% EtOAc/Hexto afford ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (2.71g, 47%). ¹H NMR (400 MHz, Chloroform-d) δ 4.18 (q, J=7.1 Hz, 2H), 1.49(s, 6H), 1.43 (s, 9H), 1.27 (t, J=7.1 Hz, 3H).

Preparation of Ethyl 2-amino-2-methylpropanoate hydrochloride

Take up ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (2.71 g,11.72 mmol) in CH₂Cl₂ (25 mL) and slowly add 4N HCl in dioxane (25 mmol)and stir at room temperature. At 1 h, the reaction was determined to becomplete by TLC. The solvents were removed under reduced pressure andthe crude was coevaporated with Et₂O two times then placed under highvacuum to afford ethyl 2-amino-2-methylpropanoate hydrochloride (2.02 g,102%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 3H), 4.18 (q, J=7.1 Hz, 2H),1.46 (s, 6H), 1.21 (t, J=7.1 Hz, 3H).

Preparation of Ethyl2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate(Compound N)

Take up phenyl dichlorophosphate (0.97 mL, 6.50 mmol) and ethyl2-amino-2-methylpropanoate hydrochloride (1.09 g, 6.50 mmol) in CH₂Cl₂(50 mL). Cool the reaction mixture to 0° C. and slowly add TEA (1.75 mL,12.45 mmol). Remove the cold bath and allow the reaction mixture to stirat room temperature. After 2 h, the addition of the amino acid wasdetermined to be complete by ³¹P NMR. Charge p-nitrophenol (0.860 g,6.17 mmol) followed by the addition of TEA (0.87, 7.69 mmol). Allow thereaction to stir at room temperature. After 2 h, the reaction wasdetermined to be complete by LCMS. The reaction was diluted with Et₂Oand the TEA*HCl salts were filtered off. The crude was concentrated andpurified by silica gel chromatography (0-50% EtOAc/Hex) to affordcompound N (1.79 g, 68%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.37-8.21 (m, 2H),7.55-7.44 (m, 2H), 7.43-7.33 (m, 2H), 7.30-7.09 (m, 3H), 6.57 (d, J=10.1Hz, 1H), 3.99 (q, J=7.1 Hz, 2H), 1.39 (s, 6H), 1.08 (t, J=7.1 Hz, 3H).³¹P NMR (162 MHz, DMSO-d₆) δ −2.87. LC/MS: t_(R)=1.65 min, MS m/z=408.97[M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet;Column: Kinetex 2.6μ XB-C18 100A, 50×3.00 mm; Solvents: Acetonitrilewith 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN,2.85 min-3.0 min 2% ACN at 1.8 mL/min.

Preparation of ethyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate(Compound 30)

Take up compound 1 (66 mg, 0.23 mmol) in NMP (2.0 mL). Cool the mixtureto 0° C. and slowly add tBuMgCl (1.0M in THF, 0.34 mL, 0.34 mmol). Allowthe reaction to stir at 0° C. for 30 min, then add a solution ofcompound N (139 mg, 0.34 mmol) dissolved in THF (1.0 mL). Remove thecold bath and place the reaction in a 50° C. preheated oil bath. After 2h, the reaction was cooled to room temperature and quenched with aceticacid and methanol. The crude was concentrated and purified by reversephase HPLC without modifier to afford compound 30 (32 mg, 25% as amixture of diastereomers). ¹H NMR (400 MHz, DMSO-d₆) δ 7.89 (m, 3H),7.31 (q, J=8.1 Hz, 2H), 7.22-7.05 (m, 3H), 6.87 (d, J=4.5, 1H), 6.80 (d,J=4.5 Hz, 1H), 6.27 (d, J=11.7, 1H), 5.81 (d, J=9.7, 1H), 5.35 (d, J=5.6Hz, 1H), 4.64 (dt, J=9.0, 5.6 Hz, 1H), 4.24 (m, 2H), 4.11 (m, 1H),4.04-3.90 (m, 3H), 1.39-1.23 (m, 6H), 1.10 (t, J=7.1, 3H). ³¹P NMR (162MHz, DMSO-d₆) δ 2.45, 2.41. LC/MS: t_(R)=1.03 min, MS m/z=561.03 [M+1];LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet;Column: Kinetex 2.6μ XB-C18 100A, 50×3.00 mm; Solvents: Acetonitrilewith 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN,2.85 min-3.0 min 2% ACN at 1.8 mL/min.

Example 34 Isopropyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate(31)

The preparation of Isopropyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoateis described below.

Preparation of Isopropyl2-((tert-butoxycarbonyl)amino)-2-methylpropanoate

Take up triphenylphosphine (6.17 g, 25.00 mmol) in THF (30 mL). Nextcharge DIAD (4.92 mL, 25.00 mmol) and stir at room temperature for 10min. Dissolve 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid(5.07 g, 25.00 mmol) in THF (20 mL) and add to the reaction mixturefollowed by the addition of isopropanol (1.91 mL, 25.00 mmol). Allow thereaction to stir at room temperature for 1 h. The solvents were removedunder reduced pressure and the crude was taken up in 1:1 Et₂O:Hexanes(120 mL). The solid triphenylphosphine oxide was filtered off and thesolvent was removed under reduced pressure. The crude was taken up inminimal CH₂Cl₂ and purified by silica gel chromatography (0-50%EtOAc/Hex) to afford isopropyl2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (4.09 g, 67%). ¹H NMR(400 MHz, Chloroform-d) δ 5.03 (p, J=6.2 Hz, 1H), 1.48 (s, 6H), 1.40 (d,J=6.2 Hz, 9H), 1.24 (d, J=6.3 Hz, 6H).

Preparation of Isopropyl 2-amino-2-methylpropanoate hydrochloride

Take up isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate(4.09 g, 16.67 mmol) in CH₂Cl₂ (50 mL) and slowly add 4N HCl in dioxane(50 mmol) and stir at room temperature. At 1 h, the reaction wasdetermined to be complete by TLC. The solvents were removed underreduced pressure and the crude was coevaporated with Et₂O two times thenplaced under high vacuum to afford isopropyl 2-amino-2-methylpropanoatehydrochloride (3.06 g, 101%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 3H),4.96 (p, J=6.2 Hz, 1H), 1.44 (s, 6H), 1.22 (d, J=6.2 Hz, 6H).

Preparation ofIsopropyl2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (Compound O)

Take up phenyl dichlorophosphate (0.83 mL, 5.58 mmol) and isopropyl2-amino-2-methylpropanoate hydrochloride (1.01 g, 5.58 mmol) in CH₂Cl₂(50 mL). Cool the reaction mixture to 0° C. and slowly add TEA (1.61 mL,11.45 mmol). Remove the cold bath and allow the reaction mixture to stirat room temperature. After 2 h, the addition of the amino acid wasdetermined to be complete by ³¹P NMR. Charge p-nitrophenol (0.74 g, 5.30mmol) followed by the addition of TEA (0.81, 5.84 mmol). Allow thereaction to stir at room temperature. After 2 h, the reaction wasdetermined to be complete by LCMS. The reaction was diluted with Et₂Oand the TEA*HCl salts were filtered off. The crude was concentrated andpurified by silica gel chromatography (0-50% EtOAc/Hex) to affordcompound O (1.45 g, 62%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.42-8.19 (m, 2H),7.55-7.43 (m, 2H), 7.39 (dd, J=8.6, 7.2 Hz, 2H), 7.30-7.12 (m, 3H), 6.53(d, J=10.1 Hz, 1H), 4.82 (hept, J=6.3 Hz, 1H), 1.38 (s, 6H), 1.09 (d,J=6.3, 6H). ³¹P NMR (162 MHz, DMSO-d₆) δ −2.84. LC/MS: t_(R)=1.73 min,MS m/z=422.92 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system:Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50×3.00 mm;Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formicacid; Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8 mL/min.

Preparation of Isopropyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate(Compound 31)

Take up compound 1 (66 mg, 0.23 mmol) in NMP (2.0 mL). Cool the mixtureto 0° C. and slowly add tBuMgCl (1.0M in THF, 0.57 mL, 0.57 mmol). Allowthe reaction to stir at 0° C. for 30 min, then add a solution ofcompound O (143 mg, 0.34 mmol) dissolved in THF (1.0 mL). Remove thecold bath and place the reaction in a 50° C. preheated oil bath. After 2h, the reaction was cooled to room temperature and was quenched withacetic acid and methanol. The crude was concentrated and purified byreverse phase HPLC without modifier to afford compound 31 (48 mg, 37% asa mixture of diastereomers). ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (m, 3H),7.30 (td, J=8.5, 7.0 Hz, 2H), 7.20-7.04 (m, 3H), 6.87 (d, J=4.5, 1H),6.80 (d, J=4.5 Hz, 1H), 6.27 (d, 6.1 Hz, 1H), 5.75 (t, J=9.1 Hz, 1H),5.34 (d, J=5.7 Hz, 1H), 4.81 (p, J=6.3 Hz, 1H), 4.71-4.50 (m, 1H), 4.23(m, 2H), 4.11 (m, 1H), 4.03-3.83 (m, 1H), 1.37-1.23 (m, 6H), 1.18-1.04(m, 6H). ³¹P NMR (162 MHz, dmso) δ 2.47, 2.43. LC/MS: t_(R)=1.08 min, MSm/z=575.06 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: ThermoLCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50×3.00 mm; Solvents:Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid;Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8 mL/min.

Example 35 (S)-2-ethylbutyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(32)

The preparation of (S)-2-ethylbutyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoateis described below.

Preparation of(3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one

(3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol(15.0 g) was combined with MTBE (60.0 mL), KBr (424.5 mg), aqueousK₂HPO₄ solution (2.5M, 14.3 mL), and TEMPO (56 mg). This mixture wascooled to about 1° C. Aqueous bleach solution (7.9% wt.) was slowlycharged in portions until complete consumption of starting material asindicated through a starch/iodide test. The layers were separated, andthe aqueous layer was extracted with MTBE. The combined organic phasewas dried over MgSO₄ and concentrated under reduced pressure to yieldthe product as a solid.

Preparation (4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine)

To a cold solution of 4-aminopyrrolo[2,1-f][1,2,4]-triazine (10.03 g;74.8 mmol) in N,N-dimethylformamide (70.27 g), N-iodosuccinimide (17.01g; 75.6 mmol) was charged in portions, while keeping the contents atabout 0° C. Upon reaction completion (about 3 h at about 0° C.), thereaction mixture was transferred into a 1 M sodium hydroxide aqueoussolution (11 g NaOH and 276 mL water) while keeping the contents atabout 20-30° C. The resulting slurry was agitated at about 22° C. for1.5 h and then filtered. The solids are rinsed with water (50 mL) anddried at about 50° C. under vacuum to yield4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine as a solid. ¹H NMR (400 MHz,DMSO-d6) δ 7.90 (s, 1H), 7.78 (br s, 2H), 6.98 (d, J=4.4 Hz, 1H), 6.82(d, J=4.4 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d6) δ 155.7, 149.1, 118.8,118.1, 104.4, 71.9. MS m/z=260.97 [M+H].

Preparation(3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol via (4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine)

To a reactor under a nitrogen atmosphere was charged iodobase 2 (81 g)and THF (1.6 LV). The resulting solution was cooled to about 5° C., andTMSCl (68 g) was charged. PhMgCl (345 mL, 1.8 M in THF) was then chargedslowly while maintaining an internal temperature at about ≤5° C. Thereaction mixture was stirred at about 0° C. for 30 min, and then cooledto about −15° C. iPrMgCl-LiCl (311 mL, 1.1 M in THF) was charged slowlywhile maintaining an internal temperature below about −12° C. Afterabout 10 minutes of stirring at about −15° C., the reaction mixture wascooled to about −20° C., and a solution of lactone 1 (130 g) in THF (400mL) was charged. The reaction mixture was then agitated at about −20° C.for about 1 h and quenched with AcOH (57 mL). The reaction mixture waswarmed to about 0° C. and adjusted to pH 7-8 with aqueous NaHCO₃ (5 wt%, 1300 mL). The reaction mixture was then diluted with EtOAc (1300 mL),and the organic and aqueous layers were separated. The organic layer waswashed with 1N HCl (1300 mL), aqueous NaHCO₃ (5 wt %, 1300 mL), andbrine (1300 mL), and then dried over anhydrous Na₂SO₄ and concentratedto dryness. Purification by silica gel column chromatography using agradient consisting of a mixture of MeOH and EtOAc afforded the product.

Preparation ((2S)-2-ethylbutyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate) (mixture ofSp and Rp)

L-Alanine 2-ethylbutyl ester hydrochloride (5.0 g, 23.84 mmol) wascombined with methylene chloride (40 mL), cooled to about −78° C., andphenyl dichlorophosphate (3.65 mL, 23.84 mmol) was added. Triethylamine(6.6 mL, 47.68 mmol) was added over about 60 min at about −78° C. andthe resulting mixture was stirred at ambient temperature for 3 h. Thereaction mixture was cooled to about 0° C. and pentafluorophenol (4.4 g,23.84 mmol) was added. Triethylamine (3.3 mL, 23.84 mmol) was added overabout 60 min. The mixture was stirred for about 3 h at ambienttemperature and concentrated under reduced pressure. The residue wasdissolved in EtOAc, washed with an aqueous sodium carbonate solutionseveral times, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography using a gradient of EtOAcand hexanes (0 to 30%). Product containing fractions were concentratedunder reduced pressure to give (2S)-2-ethylbutyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate as a solid.¹H NMR (400 MHz, Chloroform-d) δ 7.41-7.32 (m, 4H), 7.30-7.17 (m, 6H),4.24-4.16 (m, 1H), 4.13-4.03 (m, 4H), 4.01-3.89 (m, 1H), 1.59-1.42 (m,8H), 1.40-1.31 (m, 8H), 0.88 (t, J=7.5 Hz, 12H). ³¹P NMR (162 MHz,Chloroform-d) δ −1.52. ¹⁹F NMR (377 MHz, Chloroform-d) δ −153.63,−153.93 (m), −160.05 (td, J=21.9, 3.6 Hz), −162.65 (qd, J=22.4, 20.5,4.5 Hz). MS m/z=496 [M+H].

Preparation of Title Compound (mixture of Sp and Rp)

The nucleoside (29 mg, 0.1 mmol) and the phosphonamide (60 mg, 0.12mmol) and N,N-dimethylformamide (2 mL) were combined at ambienttemperature. Tert-Butyl magnesium chloride (1M in THF, 0.15 mL) wasslowly added. After about 1 h, the reaction was diluted with ethylacetate, washed with aqueous citric acid solution (5% wt.), aqueoussaturated NaHCO₃ solution and saturated brine solution. The organicphase was dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography using agradient of methanol and CH₂Cl₂ (0 to 5%). Product containing fractionswere concentrated under reduced pressure to provide the product.

Preparation of(3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbonitrile

To a mixture of(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile(5.8 g, 0.02 mol), 2,2-dimethoxypropane (11.59 mL, 0.09 mol) and acetone(145 mL) at ambient temperature was added sulfuric acid (18M, 1.44 mL).The mixture was warmed to about 45° C. After about 30 min, the mixturewas cooled to ambient temperature and sodium bicarbonate (5.8 g) andwater 5.8 mL) were added. After 15 min, the mixture was concentratedunder reduced pressure. The residue was taken up in ethyl acetate (150mL) and water (50 mL). The aqueous layer was extracted with ethylacetate (2×50 mL). The combined organic phase was dried over sodiumsulfate and concentrated under reduced pressure to give crude(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile.¹H NMR (400 MHz, CD₃OD) δ 7.84 (s, 1H), 6.93 (d, J=4.6 Hz, 1H), 6.89 (d,J=4.6 Hz, 1H), 5.40 (d, J=6.7 Hz, 1H), 5.00 (dd, J=6.7, 3.3 Hz, 1H),4.48-4.40 (m, 1H), 3.81-3.72 (m, 2H), 1.71 (s, 3H), 1.40 (s, 3H). MSm/z=332.23 [M+1].

Preparation of (2S)-2-ethylbutyl2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate

Acetonitrile (100 mL) was combined with (2S)-2-ethylbutyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)-amino)propanoate (9.6 g, 21.31mmol), the substrate alcohol (6.6 g, 0.02 mol), magnesium chloride (1.9g, 19.91 mmol) at ambient temperature. The mixture was agitated forabout 15 min and N,N-diisopropylethylamine (8.67 mL, 49.78 mmol) wasadded. After about 4 h, the reaction was diluted with ethyl acetate (100mL), cooled to about 0° C. and combined with aqueous citric acidsolution (5% wt., 100 mL). The organic phase was washed with aqueouscitric acid solution (5% wt., 100 mL) and aqueous saturated ammoniumchloride solution (40 mL), aqueous potassium carbonate solution (10%wt., 2×100 mL), and aqueous saturated brine solution (100 mL). Theorganic phase was dried with sodium sulfate and concentrated underreduced pressure to provide crude product. ¹H NMR (400 MHz, CD₃OD) δ7.86 (s, 1H), 7.31-7.22 (m, 2H), 7.17-7.09 (m, 3H), 6.93-6.84 (m, 2H),5.34 (d, J=6.7 Hz, 1H), 4.98 (dd, J=6.6, 3.5 Hz, 1H), 4.59-4.50 (m, 1H),4.36-4.22 (m, 2H), 4.02 (dd, J=10.9, 5.7 Hz, 1H), 3.91 (dd, J=10.9, 5.7Hz, 1H), 3.83 (dq, J=9.7, 7.1 Hz, 1H), 1.70 (s, 3H), 1.50-1.41 (m, 1H),1.39 (s, 3H), 1.36-1.21 (m, 7H), 0.86 (t, J=7.4 Hz, 6H). MS m/z=643.21[M+1].

Preparation of (S)-2-ethylbutyl2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(Compound 32)

The crude acetonide (12.85 g) was combined with tetrahydrofuran (50 mL)and concentrated under reduced pressure. The residue was taken up intetrahydrofuran (100 mL), cooled to about 0° C. and concentrated HCl (20mL) was slowly added. The mixture was allowed to warm to ambienttemperature. After consumption of the starting acetonide as indicated byHPLC analysis, water (100 mL) was added followed by aqueous saturatedsodium bicarbonate solution (200 mL). The mixture was extracted withethyl acetate (100 mL), the organic phase washed with aqueous saturatedbrine solution (50 mL), dried over sodium sulfated and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography using a gradient of methanol and ethyl acetate (0 to20%). Product containing fractions were concentrated under reducedpressure to provide the product.

B. Antiviral Activity

Another aspect of the invention relates to methods of inhibiting viralinfections, comprising the step of treating a sample or subjectsuspected of needing such inhibition with a composition of theinvention.

Within the context of the invention samples suspected of containing avirus include natural or man-made materials such as living organisms;tissue or cell cultures; biological samples such as biological materialsamples (blood, serum, urine, cerebrospinal fluid, tears, sputum,saliva, tissue samples, and the like); laboratory samples; food, water,or air samples; bioproduct samples such as extracts of cells,particularly recombinant cells synthesizing a desired glycoprotein; andthe like. Typically the sample will be suspected of containing anorganism which induces a viral infection, frequently a pathogenicorganism such as a tumor virus. Samples can be contained in any mediumincluding water and organic solvent\water mixtures. Samples includeliving organisms such as humans, and man made materials such as cellcultures.

If desired, the anti-virus activity of a compound of the invention afterapplication of the composition can be observed by any method includingdirect and indirect methods of detecting such activity. Quantitative,qualitative, and semiquantitative methods of determining such activityare all contemplated. Typically one of the screening methods describedabove are applied, however, any other method such as observation of thephysiological properties of a living organism are also applicable.

The antiviral activity of a compound of the invention can be measuredusing standard screening protocols that are known. For example, theantiviral activity of a compound can be measured using the followinggeneral protocols:

MOI Incu- Cell Plate Cell (pfu/ bation Virus Line Format Number cell)(Days) Read Out Values Junin Vero 96 20,000 0.003 5 to 7 Neutral EC50red staining Junin HeLa 384 or 96 2,000 0.3 2 HCS Lassa HeLa 384 or 962,000 0.3 2 HCS HCS: High content imaging HeLa: Hela epithelial cell(cervical carcinoma)

Example 36 Lassa Virus and Junin Virus Antiviral Activity andCytotoxicity Assays

Antiviral activity of Compound 1, Compound 9, and Compound 32 wasmeasured against Lassa virus (LASV) and Junin virus (JUNV). All studiesconducted with wild-type virus were performed in biosafety level-4containment (BSL-4) at the US Army Medical Research Institute forInfectious Diseases (USAMRIID) Antiviral Assays conducted with anattenuated strain of JUNV were conducted at Utah State University in aBSL-2 laboratory. Lassa virus antiviral assays were conducted HeLacells. Junin virus antiviral assays were conducted in Vero and HeLacells.

Antiviral assays were conducted in 384 or 96 well plates in BSL-4containment using a high content imaging system to quantify virusantigen production as a measure of virus replication. A “no virus”control (Column 2) and a “1% DMSO” control (Column 3) were included oneach plate to determine the 0% and 100% virus replication signal,respectively. The primary antibodies used for detection of viralantigens were mm L52-161-6 anti-GP; LASV and mm Y-GQC03_BF11 anti-GP;JUNV and DyLight 488 anti-mouse-IgG was used as the secondary detectionantibody. The primary antibody was diluted 1000-fold in blocking buffer(1×PBS with 3% BSA) and added to each well of the assay plate. The assayplates were incubated for 60 minutes at room temperature. The primaryantibody was removed and the cells were washed 3 times with 1×PBS. Thesecondary antibody was diluted 1000-fold in blocking buffer and wasadded to each well in the assay plate. The assay plates were incubatedfor 60 minutes at room temperature. Nuclei were stained using Draq5(Biostatus, Shepshed Leicestershire, UK, Cat #DR05500) diluted in 1×PBS.Cell images were acquired using Perkin Elmer Opera confocal microscope(Perkin Elmer, Waltham, Mass.) using 10× air objective to collect fiveimages per well. Virus-specific antigen was quantified by measuringfluorescence emission at a 488 nm wavelength and the nuclei werequantified by measuring fluorescence emission at a 640 nm wavelength.The Z′ values for all antiviral assays were >0.3.

The percentage inhibition was calculated for each tested concentrationrelative to the 0% and 100% inhibition controls and the EC₅₀ value foreach compound was determined by non-linear regression as the effectiveconcentration of compound that inhibited virus replication by 50%.

Example 37 Junin Virus Assay—Vero

Vero or Vero E6 cells were seeded in 96 well plates at 20,000 cells perwell in 100 uL of MEM+2% FBS. Compounds diluted in DMSO were mixed with120 uL of MEM+2% FBS. 100 uL of each test compound are transferred to 2wells of a 96-well plate. 20 uL of virus solution in MEM+20% FBS areadded so that final test concentrations are 47, 4.7, 0.47, 0.047 uM andthe multiplicity of infection was 0.003 pfu/cell. Test plates wereincubated until untreated virus controls approached maximum cytopathiceffects (CPE) (5 to 7 days). Plates are then stained with neutral reddye for 2 hrs then eluted in Citrate/Ethanol buffer and read on aspectrophotometer at 540 nm. EC50 value is calculated by regressionanalysis as the concentration of test compound required to reduceviral-induced CPE by 50% measured by neutral red staining.

Example 38 Junin Virus Assay—HeLa

HeLa cells were seeded at 2000 cells per well in a 384 well plate andcompounds were added to the assay plates as described in section 3.2.1.Assay plates were transferred to the BSL-4 suite and infected with 0.3pfu per cell JUNV which resulted in ˜50% of the cells expressing virusantigen in a 48 h period. The assay plates were incubated for 48 h andvirus replication was quantified by immuno-staining using antibodiesthat recognized the viral glycoproteins.

Example 39 Lassa Virus Assay

HeLa cells were seeded at 2000 cells per well in a 384 well plate andcompounds were added to the assay plates as described in section 3.2.1.Assay plates were transferred to the BSL-4 suite and infected with 0.1pfu per cell LASV which resulted in >60% of the cells expressing virusantigen in a 48h period. The assay plates were incubated for 48 handvirus replication was quantified by immuno-staining using antibodiesthat recognized the viral glycoproteins.

TABLE 2 Lassa Virus and Junin Virus antiviral assays Table 2: In VitroAntiviral Activity of Compounds 1, 9, and 32 against arenaviruses EC₅₀(μM) EC₉₀ (μM) Assay HCS HCS HCS HCS Virus Junin Junin Lassa Junin JuninLassa Cell Line Vero HeLa HeLa Vero HeLa HeLa Compound 1 >47, 19 N.D.N.D. >47 N.D. N.D. Compound 9 >47 0.49 2.0 >47 1.25 4.21 Compound 32N.D. 0.47 1.65 N.D. 1.26 3.31 N.D. not determined

JUNV=Junin virus, LASV=Lassa virus

Example 40 MERS-CoV and SARS-CoV Antiviral Activity and CytotoxicityAssays

Antiviral activity of Compound 9 and Compound 32 was measured againstMERS virus (MERS-CoV) and SARS virus (SARS-CoV).

Antiviral assays were conducted at USAMRIID and the University of NorthCarolina at Chapel Hill.

Example 41 MERS-CoV Antiviral Assay (USAMRIID)

Vero E6 cells seeded in 384-well plates and serial dilutions of Compound32 or Compound 9 were added to the assay plates by direct titrationusing an HP D300 Digital Dispenser (Hewlett-Packard, Palo Alto, Calif.).The plates were transferred to the BSL-4 suite and infected withMERS-CoV (Strain Jordan N₃) at a multiplicity of infection of 0.5 plaqueforming unit (pfu) per cell. The infected cultures were incubated for 48hours. The level of virus replication in compound-treated and controlvehicle-treated cultures was determined by quantifying the level ofvirus-specific antigen following immuno-staining with antibody againstthe MERS-CoV spike (S) protein. The primary antibody (40069-RP02rb-HCoV-EMC/2012 spike(S) protein) was diluted 1000-fold in blockingbuffer (1× phosphate buffered saline (PBS) with 3% BSA) and added toeach well of the assay plate. The assay plates were incubated for 60minutes at room temperature. The primary antibody was removed and thecells were washed 3 times with 1× PBS. The secondary detection antibodywas an anti-rabbit IgG conjugated with Dylight488 (Thermo FisherScientific, Waltham, Mass., Cat #405310). The secondary antibody wasdiluted 1000-fold in blocking buffer and was added to each well in theassay plate. The assay plates were incubated for 60 minutes at roomtemperature. Nuclei were stained using Draq5 (Biostatus, ShepshedLeicestershire, UK, Cat #DR05500) diluted in 1× PBS. The cells werecounter-stained with CellMask Deep Red (Thermo Fisher Scientific,Waltham, Mass., Cat #C10046) to enhance detection of the cytoplasmcompartment. Cell images were acquired using Perkin Elmer Opera confocalmicroscope (Perkin Elmer, Waltham, Mass.) using 10× air objective tocollect 5 images per well. Virus-specific antigen was quantified bymeasuring fluorescence emission at a 488 nm wavelength and the nucleiwere quantified by measuring fluorescence emission at a 640 nmwavelength. High content image analysis was performed to quantify thepercent of infected cells and cell viability. Analysis of dose responseto determine EC50 values was performed using GeneData Screener softwareapplying Levenberg-Marquardt algorithm for curve fitting strategy.

Example 42 MERS-CoV and SARS-CoV Antiviral Assay

HAE cell cultures isolated from lung tissue were cultured for 6 weeks atthe air liquid interface to promote differentiation. The apical surfacesof the HAE cultures were washed at 24 h and 1 h prior to infection with1× PBS for >1 hour at 37 oC. Recombinant MERS-CoV expressing redfluorescent protein (MERS-CoV RFP) and SARS-CoV expressing greenfluorescent protein (SARS-CoV GFP) were used to apically infect thedifferentiated HAE cultures at a multiplicity of infection of 0.1 pfuper cell. To infect the HAE cultures, apical washes were removed, viralinoculum was added, and inoculated cultures were incubated at 37° C. for2.5 hours. The inoculum was removed, and the apical surfaces of the HAEcultures were washed 3 times with 500 μL of 1× PBS to remove residualvirus. Five 3-fold serial dilutions of Compound 9 starting at 10 μM wereprepared in triplicate and added to HAE ALI media on the basolateralside of the culture approximately 30 minutes prior to infection. Virusreplication was assessed by fluorescence imaging of cell culturesfollowing a 48-hour incubation. In addition, virus replication wasquantified by measuring the production of infectious virus in HAE apicalwashes by plaque assay on Vero cell monolayers and by quantifying viralRNA production from total cell RNA by real-time PCR assay.

TABLE 3 MERS antiviral assays Table 3: In Vitro Antiviral Activity ofCompound 32 against coronaviruses Assay EC₅₀ (μM) Virus MERS-CoV CellLine Vero Compound 9 0.46 Compound 32 0.58 MERS = Middle EastRespiratory Syndrome

Example 43 MERS-CoV and SARS-CoV Real-Time PCR Assay

At 48 hours post-infection, primary HAE cultures from the antiviralassay described above were harvested in 500 μL TRIzol. RNA was purifiedusing a Direct-zol RNA MiniPrep kit (Zymo Research Corporation, Irvine,Calif., USA). First-strand cDNA was generated for each sample usingSuperScript III (Life Technologies, Grand Island, N.Y., USA) withincubation at 55° C. Following first-strand cDNA generation, ORF1(genome RNA) and ORF8 or ORF9 (MERS-CoV and SARS-CoV subgenomic RNA,respectively) were quantified by real-time PCR using the followingprimers: MERS-CoV: Leader Forward (5′-GAA TAG CTT GGC TAT CTC AC-3′ SEQID NO: 1), ORF1 Reverse (5′-CAC AAT CCC ACC AGA CAA 3′ SEQ ID NO: 2),ORF8 Reverse (5′-TTG TTA TCG GCA AAG GAA AC-3′ SEQ ID NO: 3); andSARS-CoV: Leader Forward (5′-AGC CAA CCA ACC TCG ATC TCT TGT-3′ SEQ IDNO: 4), ORF1 Reverse (5′-TGA CAC CAA GAA CAA GGC TCT CCA-3′ SEQ ID NO:5), ORF9 Reverse (5′-ATT GGT GTT GAT TGG AAC GCC CTG-3′ SEQ ID NO: 6).Reads were normalized to GAPDH using the following primers: GAPDHForward (5′-TGC ACC ACC AAC TGC TTA GC-3′ SEQ ID NO: 7) and GAPDHReverse (5′-GGC ATG GAC TGT GGT CAT GAG-3′ SEQ ID NO: 8). Results areexpressed as log 10 fold changes in viral ORF1 and ORF8-encoding RNA(MERS-CoV)/and ORF9-encoding RNA (SARS-CoV) copy number in treatedversus untreated cells using the ΔΔCt method {10431}.

Example 44 In Fitro Efficacy in Calu-3 2B4 Cells

At 48 hrs prior to infection, Calu-3 2B4 cells were plated in a 96-wellblack walled clear bottom plate at 5×10⁴ cells/well. 24-hr prior toinfection, culture medium was replaced. A 20 mM stock of Compound 32 wasserially diluted in 100% DMSO in 3-fold increments to obtain a ten-pointdilution series. MERS-nLUC was diluted in DMEM 10% FBS, and 1%antibiotics/antimycin to achieve a multiplicity of infection (MOI) of0.08. Cells were infected in triplicate per drug dilution for 1 hr afterwhich, virus was aspirated, cultures were rinsed once and fresh mediumcontaining drug or vehicle was added. At 48 hrs post infection, virusreplication was quantitated on a Spectramax (Molecular Devices) platereader via nano-luciferase assay (Promega) according to themanufacturer's protocol. For our 100% inhibition control, dilutedMERS-nLUC was exposed to short-wave UV light (LLC, Upland, Calif.) for 6minutes to inhibit the ability of the virus to replicate. For our 0%inhibition control, cells were infected in the presence of vehicle. DMSOwas kept constant in all conditions at 0.05% by volume (v/v). Valuesfrom triplicate wells per condition were averaged and compared tocontrols to generate a percent inhibition value for each drug dilution.The EC₅₀ value was defined as the concentration at which there was a 50%decrease in viral replication. Data were analyzed using GraphPad Prism6.0 (La Jolla, Calif.). The EC₅₀ and CC₅₀ values were calculated bynon-linear regression analysis using the dose-response (variable slope)equation (four parameter logistic equation):Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log EC₅₀-X)*HillSlope)). The “Bottom” and “Top” values are defined by the minimum andmaximum Y values. Hill slope is a parameter used to define the steepnessof a dose-response curve. EC₅₀ and CC₅₀ values were calculated as anaverage of two to four independent experiments.

TABLE 4 Antiviral activity of Compound 1 and Compound 32 againstMERS-CoV and SARS-CoV and cytotoxicity. EC₅₀ (μM)¹ MERS SARS CC₅₀ (μM)Compound 1 0.46 (HAE) 0.22 (HAE) >100 (HAE) — (Calu-3) — (Calu-3) >100(Calu-3) Compound 32 0.074 (HAE) 0.069 (HAE) >10 (HAE) 0.03 (Calu-3)0.01 (Calu-3) >10 (Calu-3) ¹All values are averages from >3 independentexperiments. HAE = Human airway epithelial cell. Calu-3 = human lungepithelial cell line Calu-3 (Calu3-2B4). HAE studies were done fromthree donors.

Example 45 Evaluation of Subcutaneous Compound 32 Against Severe AcuteRespiratory Syndrome Coronavirus (SARS-CoV) in Esterase Deficient(Ces1c−/−) Mice

Male and female mice (25-28 week) genetically deleted forcarboxylesterase 1C (Ces1c−/−) (Jackson Laboratories stock 014096). The(Ces1c−/−) mice were used since rodents express high levels ofcarboxylesterase activity in plasma relative to other animal speciesreducing the plasma half-life of Compound 32. Genetic deletion ofcarboxylesterase 1C improved the plasma stability of Compound 32generating pharmacokinetic profiles similar to those observed in humansand other animal species.

The study design is captured in Table 4. Efficacy studies were performedin an animal biosafety level 3 (ABSL3) facility. All work was conductedunder protocols approved by the Institutional Animal Care and UseCommittee at UNC Chapel Hill according to guidelines set by theAssociation for the Assessment and Accreditation of Laboratory AnimalCare (AAALAC) and the United States Department of Agriculture (USDA).

TABLE 4 Experimental Design (Subcutaneous Injection) Compound #Males/ 32Dose Timing and Chal- Group #Females Treatment (mg/kg) Duration lenge 16/6 Vehicle 0 Twice Daily, SARS- D −1 to D 5 CoV 2 4/4 Compound 25 TwiceDaily, 32 in D −1 to D 5 vehicle 3 6/6 Compound 50 Once Daily, 32 in D−1 to D 5 vehicle 4 1/2 Vehicle 0 Twice Daily, No D −1 to D 5 virus 52/1 Compound 25 Twice Daily, 32 in D −1 to D 5 vehicle

Groups 1 (vehicle), Group 2 (Compound 32 BID 25 mg/kg), and Group 3(Compound 32 QD 50 mg/kg) were anaesthetized with ketamine/xylazineexposed to 10⁴ pfu of SARS-CoV/50 ul via the intranasal route. Group 4(Vehicle) and Group 5 (Compound 32 BID 25 mg/kg) remained uninfected andwere used as controls for whole body plethysmography evaluations.Vehicle comprised 12% sulfobutylether-β-cyclodextin in water (withHCl/NaOH) at pH 5.0). On day 0, animals were exposed to virus. On days 2and 5 post infection, groups of animals were euthanized by isofluoraneoverdose and the large left lobe of the lung was placed in a 2 mL screwcap tube with 1 mL DPBS with glass beads and frozen at −80° C. untilanalyzed by plaque assay. The inferior right lobe was placed in 10%buffered formalin and stored at 4° C. until histological analysis.

Changes in lung function were determined by whole body plethysmography(WBP, Buxco lung function testing system, Data Sciences International).After a 30-minute acclimation in the plethysmograph chamber, 11respiratory responses and several quality control metrics werecontinually measured every 2-second for 5 minutes for a total of 150data points. Mean values for each parameter were determined within DSIFinepoint software.

Histological analysis was performed on formalin fixed samples andparaffin embedded tissues with 5 μm. To assess lung pathology, sectionswere stained with hematoxylin and eosin. Viral antigen in the lung wasstained using polyclonal anti-nucleocapsid antibody (Imgenex). Slideswere blinded to the evaluator and assessed for virus associated lungpathology as well as spatial location and prevalence of viral antigen.Images were captured using an Olympus BX41 microscope equipped with anOlympus DP71 camera.

Viral plaque assay was used to quantify infectious virus from frozenlung tissue. Vero E6 cells were seeded in 6-well plates at 5×10⁵cells/well. Lung tissue was thawed, homogenized via Roche Magnalyzer,and the tissue suspension was serially diluted and the dilutions used toinfect the Vero E6 cells. At 72 h post-infection, the plates were fixedand stained and the number of plaques quantified by visual inspection.

The primary endpoint for this study was viral load in lung tissue at Day5 post-infection. Additional endpoints included changes in animal bodyweight and lung function. Animal body weight was recorded daily for theduration of the in-life phase. On day −1, 1, 2, 3, and 5 afterinoculation, whole body plethysmography was performed to assess lungfunction. On Day 5, a scheduled necropsy was performed on all remaininganimals; gross lung pathology was evaluated by a board-certifiedveterinary pathologist. Lung tissue was collected for histopathologicaland virological analysis.

Body Weight and Viral Load: Changes in body weight and tissue viral loadfor each study group at Day 5 are shown in FIG. 1, FIG. 2A and FIG. 2B.As shown in FIG. 1, animals treated with Compound 32 displayed noevidence of weight loss associated with SARS-CoV infection compared tovehicle-treated animals. Infectious virus was measured in lung tissuecollected at necropsy by plaque assay. As shown in FIG. 2A and FIG. 2B,infectious virus was significantly decreased in Compound 32-treatedanimals at Day 2 and Day 5 post-infection relative to vehicle-treatedanimals. These data suggest that Compound 32 reduces replication ofSARS-CoV in the lung.

Lung Function Measurements: The effect of Compound 32 treatment onpulmonary function in SARS-CoV infected mice was evaluated by whole bodyplethysmography (WBP) (FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, andFIG. 3F). WBP showed an increase in Penh values in vehicle treated micesuggesting that virus replication in the lung increased airwayresistance. In animals treated with either 25 mg/kg of Compound 32 twiceper day or 50 mg/kg of Compound 32 once per day, Penh values were lowercompared to vehicle-treated animals and were more similar tomock-infected animals.

In vehicle-treated mice infected with SARS-CoV the length of time torelease a breath (Expiration Time) or time between breaths (EndExpiratory Pause) measured by WBP increased indicating laboredbreathing. As shown in FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, andFIG. 3F, these breathing parameters were reduced in Compound 32-treatedanimals approaching values obtained from mock-infected animals.

Example 46 A Blinded, Randomized, Vehicle-Controlled Evaluation ofIntravenous Compound 32 Against Middle East Respiratory SyndromeCoronavirus (MERS-CoV) in Rhesus Monkeys

MERS-CoV isolate HCoV-EMC/2012 was used for the challenge virus at theTest Facility. MERS-CoV isolate HCoV-EMC/2012 was provided by theViroscience Laboratory, Erasmus Medical Center, Rotterdam, TheNetherlands, and propagated in VeroE6 cells in DMEM (Sigma) supplementedwith 2% (vol/vol) FCS (Logan), 1 mM L-glutamine (Lonza), 50 U/mLpenicillin, and 50 μg/mL streptomycin (Gibco). Experimentally naïve malerhesus monkeys were randomly assigned to treatment groups and balancedby body weight.

The study design is captured in Table 5.

TABLE 5 Experimental Design (Intervenous) Compound #Males/ 32 DoseTiming and Chal- Group #Females Treatment (mg/kg) Duration* lenge 1 6/0Vehicle 0 Once Daily, MERS- D −1 to D 6 CoV 2 6/0 Compound 10 OnceDaily, 32 in D −1 to D 6 vehicle

All animals were exposed to a target dose of 7×10⁶ plaque forming unitsMERS-CoV virus diluted in 0.9% sodium chloride for inoculation. Theanimals were inoculated by multiple routes that included intranasal,ocular, and intratrachial administration. The day on which animals werechallenged was designated as Day 0.

Methods to control bias included experimental blinding. Specifically,study personnel who administered Compound 32 or vehicle treatments orroutinely evaluated animal health were experimentally blinded to thegroup assignment of all animals for the duration of the in-life phase.Unblinded personnel, who were not responsible for evaluating animalhealth, prepared individual doses from bulk ready-to-use formulationsprovided by the Sponsor. Vehicle and Compound 32 formulations wereidentical in physical appearance.

In Groups 1 and 2, once-daily vehicle treatment was administered for 7days beginning on Day −1 (one day prior to virus exposure). Each dose ofCompound 32 or vehicle was administered as a single bolus slow IVinjection in the saphenous vein at a volume of 2.0 mL/kg body weightover the course of 1 to 2 min. Doses were administered to animalsanesthetized using IM injection of a solution containing ketamine (100mg/mL) and acepromazine (10 mg/mL) at a volume of 0.1 mL/kg body weight.The weight of each animal was obtained on Day −7, and these weights wereused for dose volume determination for all administered doses ofCompound 32 or vehicle.

The primary endpoint for this study was viral load in lung tissue at Day6 post-infection. Animal health was monitored at least twice daily forthe duration of the in-life phase and clinical disease signs wererecorded. On day −7, 0, 1, 3, 5 and 6 after inoculation, clinical examswere performed on all animals to determine bodyweight, body temperature,respirations/minute (under anesthesia), and to collect x-rays, nose andthroat swabs. Whole blood and serum were collected for hematology,biochemistry and cytokine analysis. On Day 6, a scheduled necropsy wasperformed on all animals; gross lung pathology was scored (as % of lunglobe affected by gross lesions) by a board-certified veterinarypathologist and lung weight was recorded to determine the lungweight/body weight ratio. Nineteen tissues were collected forhistopathological and virological analysis

Disease signs in vehicle-treated animals were attributed to MERS-COVinfection. Cumulative clinical scores were notably higher invehicle-treated animals compared to Compound 32-treated animals. Thesedisease symptoms were less pronounced in the Compound 32-treatedanimals.

Body Weight and Viral Load: Changes in body weight, temperature andrespiration are shown in FIG. 4A, FIG. 4B, and FIG. 4C. The body weightand body temperature did not change appreciably during the course of theinfection in the presence or absence of Compound 32 treatment.Respiration rates increased over the course of infection and tended tobe higher at Day 6 in vehicle-treated animals compared to Compound32-treated animals.

Tissue Viral Load: Viral RNA was measured in lung tissue and otherorgans collected at necropsy. Changes in tissue viral RNA concentrationsfor each study group at Day 6 are shown in FIG. 5. Virus was detected inall respiratory tract tissues in vehicle-treated animals. Viral RNA inthe respiratory tract was significantly reduced in Compound 32-treatedanimals. Viral RNA was below the limit of detection in treated anduntreated animals in the liver, spleen, kidney and bladder tissue. ViralRNA was detected in all animals in the mediastinal lymph node, but inonly one vehicle-treated animal in the mandibular lymph node.

Virus was detected in nose swabs and throat swabs at Day 1, 3, 5 and 6post-infection There was no difference in viral load betweenvehicle-treated and Compound 32-treated animals. Viral RNA was detectedin one vehicle-treated animal in the urine collected at Day 6. Thechanges in white blood cell counts, neutrophils and lymphocytes areshown in FIG. 5.

All publications, patents, and patent documents cited herein above areincorporated by reference herein, as though individually incorporated byreference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, one skilled in the artwill understand that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method for treating Coronaviridae infection ina human in need thereof, comprising administering a therapeuticallyeffective amount of a compound of Formula III:

or a pharmaceutically acceptable salt thereof; wherein R² and R³ areeach OR^(a); R⁶ is CN; R⁷ is —(C═O)R¹¹ or

R⁸ is NH₂; R⁹ is H; each occurrence of R^(a) is independently H or—(C═O)R; R¹¹ is H or (C₁-C₈)alkyl which is optionally substituted byNH₂; each occurrence of R is independently H or (C₁-C₈)alkyl which isoptionally substituted by NH₂; R^(f) is H or (C₁-C₈)alkyl; and one ormore hydrogens attached to a carbon of the compound are optionallyreplaced by deuterium.
 2. The method of claim 1, wherein R⁷ is


3. The method of claim 2, wherein R^(f) is selected from the groupconsisting of methyl, ethyl, n-propyl, i-propyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, and2-methyl-1-butyl.
 4. The method of claim 1, wherein R² and R³ are eachOH.
 5. The method of claim 4, wherein R⁷ is —(C═O)R¹¹ and R¹¹ isselected from the group consisting of methyl, ethyl, n-propyl, i-propyl,1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 2-pentyl,3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, and2-methyl-1-butyl, each of which is optionally substituted by NH₂.
 6. Themethod of claim 5, wherein R¹¹ is —CH(CH₃)₂.
 7. The method of claim 5,wherein R¹¹ is —CH(NH₂)CH(CH₃)₂.
 8. The method of claim 5, wherein oneor more hydrogens attached to a carbon of the compound are replaced bydeuterium.
 9. The method of claim 6, wherein one or more hydrogensattached to a carbon of the compound are replaced by deuterium.
 10. Themethod of claim 7, wherein one or more hydrogens attached to a carbon ofthe compound are replaced by deuterium.
 11. The method of claim 1,wherein R² and R³ are each —O(C═O)(C₁-C₈)alkyl, wherein the (C₁-C₈)alkylgroups of R² and R³ are each optionally substituted with NH₂.
 12. Themethod of claim 11, wherein the (C₁-C₈)alkyl groups of R² and R³ areeach selected from the group consisting of methyl, ethyl, n-propyl,i-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,3-methyl-1-butyl, and 2-methyl-1-butyl, each of which is optionallysubstituted with NH₂.
 13. The method of claim 12, wherein each of R² andR³ is selected from the group consisting of —O(C═O)—CH(CH₃)₂ and—O(C═O)—CH(NH₂)CH(CH₃)₂.
 14. The method of claim 13, wherein R² and R³are —O(C═O)—CH(NH₂)CH(CH₃)₂ and —O(C═O)—CH(CH₃)₂, respectively;—O(C═O)—CH(CH₃)₂ and —O(C═O)—CH(NH₂)CH(CH₃)₂, respectively; or—O(C═O)—CH(NH₂)CH(CH₃)₂ and —O(C═O)—CH(NH₂)CH(CH₃)₂, respectively. 15.The method of claim 13, wherein R² and R³ are each —O(C═O)—CH(CH₃)₂. 16.The method of claim 11, wherein R⁷ is —(C═O)R¹¹ and R¹¹ is —(C₁-C₈)alkyloptionally substituted by NH₂.
 17. The method of claim 16, wherein R¹¹is selected from the group consisting of methyl, ethyl, n-propyl,i-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,3-methyl-1-butyl, and 2-methyl-1-butyl, each of which is optionallysubstituted by NH₂.
 18. The method of claim 17, wherein R¹¹ is—CH(CH₃)₂.
 19. The method of claim 17, wherein R¹¹ is —CH(NH₂)CH(CH₃)₂.20. The method of claim 11, wherein R² is —O(C═O)—CH(CH₃)₂, R³ is—O(C═O)—CH(CH₃)₂, R⁷ is —(C═O)R¹¹, and R¹¹ is —CH(CH₃)₂.
 21. The methodof claim 18, wherein one or more hydrogens attached to a carbon of thecompound are replaced by deuterium.
 22. The method of claim 19, whereinone or more hydrogens attached to a carbon of the compound are replacedby deuterium.
 23. The method of claim 20, wherein one or more hydrogensattached to a carbon of the compound are replaced by deuterium.
 24. Themethod of claim 20, wherein one hydrogen attached to a carbon of thecompound is replaced by deuterium.
 25. The method of claim 1, wherein R²is OH and R³ is —O(C═O)(C₁-C₈)alkyl, wherein the (C₁-C₈)alkyl of R³ isoptionally substituted by NH₂.
 26. The method of claim 25, wherein the(C₁-C₈)alkyl of R³ is selected from the group consisting of methyl,ethyl, n-propyl, i-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl,2-methyl-2-propyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, and 2-methyl-1-butyl, each of whichis optionally substituted by NH₂.
 27. The method of claim 26, wherein R³is —O(C═O)—CH(CH₃)₂.
 28. The method of claim 26, wherein R³ is—O(C═O)—CH(NH₂)CH(CH₃)₂.
 29. The method of claim 25, wherein R⁷ is—(C═O)R¹¹ and R¹¹ is selected from the group consisting of methyl,ethyl, n-propyl, i-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl,2-methyl-2-propyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, and 2-methyl-1-butyl, each of whichis optionally substituted by NH₂.
 30. The method of claim 29, whereinR¹¹ is —CH(CH₃)₂.
 31. The method of claim 29, wherein R¹¹ is—CH(NH₂)CH(CH₃)₂.
 32. The method of claim 30, wherein one or morehydrogens attached to a carbon of the compound are replaced bydeuterium.
 33. The method of claim 31, wherein one or more hydrogensattached to a carbon of the compound are replaced by deuterium.
 34. Themethod of claim 1, wherein R³ is OH and R² is —O(C═O)(C₁-C₈)alkyl,wherein the (C₁-C₈)alkyl of R² is optionally substituted by NH₂.
 35. Themethod of claim 34, wherein the (C₁-C₈)alkyl of R² is selected from thegroup consisting of methyl, ethyl, n-propyl, i-propyl, or 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, and2-methyl-1-butyl, each of which is optionally substituted by NH₂. 36.The method of claim 35, wherein R² is —O(C═O)—CH(CH₃)₂.
 37. The methodof claim 35, wherein R² is —O(C═O)—CH(NH₂)CH(CH₃)₂.
 38. The method ofclaim 34, wherein R⁷ is —(C═O)R¹¹ and R¹¹ is selected from the groupconsisting of methyl, ethyl, n-propyl, i-propyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, and2-methyl-1-butyl, each of which is optionally substituted by NH₂. 39.The method of claim 38, wherein R¹¹is —CH(CH₃)₂.
 40. The method of claim38, wherein R¹¹ is —CH(NH₂)CH(CH₃)₂.
 41. The method of claim 39, whereinone or more hydrogens attached to a carbon of the compound are replacedby deuterium.
 42. The method of claim 40, wherein one or more hydrogensattached to a carbon of the compound are replaced by deuterium.
 43. Themethod of claim 1, wherein the Coronaviridae infection is caused by aSARS virus.
 44. The method of claim 1, wherein the Coronaviridaeinfection is caused by a Coronaviridae virus selected from the groupconsisting of SARS, MERS, 229E, NL63, OC43, and HKU1.
 45. The method ofclaim 1, wherein one or more hydrogens attached to a carbon of thecompound are replaced by deuterium.